E7 regulation of p21 cip1 through akt

ABSTRACT

Disclosed are compositions and methods for inhibiting E7 effects on Akt, such as preventing the nuclear localization of p21 Cip1 .

This application claims benefit of U.S. Provisional Application No.60/374,245, filed May 19, 2002, which is hereby incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

p21^(Cip1) is a potent inhibitor of cyclin-dependent kinase-2 (CDK2)activity. The human papillomavirus E7 oncoprotein abrogatesp21^(Cip1)-mediated G1-arrest in response to anti-proliferative signals.The mechanism by which E7 antagonizes p21^(Cip1) function in vivo isunclear. Disclosed is the use of an engineered conditional Raf kinasethat induces a p21^(Cip1)-mediated cell cycle arrest along with variousother molecules.

Disclosed herein E7 abrogates Raf-associated arrest and preventsinhibition of cyclin E-CDK2 activity without disrupting Raf induction ofp21^(Cip1). E7 neither interacts with p21^(Cip1) nor derepressesp21^(Cip1)-associated CDK2 activity, but instead reduces the associationbetween p21^(Cip1) and cyclin E-CDK2 complexes. Disclosed herein it isshown that Raf down-regulates steady-state levels of Akt, a regulator ofp21^(Cip1) localization, leading to loss of p21^(Cip1) phosphorylationand accumulation of nuclear p21^(Cip1). It is also shown herein that E7disrupts the effects of Raf on Akt activity and prevents p21^(Cip1)nuclear accumulation. It is also diclosed herein that maintenance of Aktactivity is necessary and sufficient to bypass Raf arrest.

SUMMARY OF THE INVENTION

In accordance with the purposes of this invention, as embodied andbroadly described herein, this invention, in one aspect, relates tocompositions and methods for identifying inhibitors of E7 cellproliferation activity.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain theprinciples of the invention.

FIG. 1 shows that E7 abrogates RafAR-induced arrest. (A) NIH3T3 cellsstably expressing the RafAR fusion protein were infected withamphotrophic retroviruses expressing HPV-16 E7 (E7) or empty vector(Babe). Lysates were prepared and 100 μg of protein was resolved by 15%SDS-PAGE, transferred to nitrocellulose membrane, and probed with amonoclonal antibody directed against HPV-16 E7. (B and C) Pools of Babe-or E7-expressing cells growing in DMEM plus 10% NCS were treated with0.02% ethanol (−) or 1.0 μM R1881 (+) for 30 hours and pulsed with BrdUfor 30 minutes. Cells were trypsinized, fixed in 70% ethanol, andstained with propidium iodide (PI) for detection of total DNA content(B) and with α-BrdU-FITC for detection of DNA synthesis (C).

FIG. 2 shows that E7 prevents p21Cip1-mediated inhibition of cyclinE-CDK2 activity. (A) Expression levels of proteins involved inregulating the G1-S transition were examined during RafAR activation inthe presence or absence of E7. Lysates were prepared from Babe- orE7-expressing cells treated with 0.02% ethanol (−) or 1.0 μM R1881 (+)for 30 hours, and 30 μg of protein was resolved by 12% SDS-PAGE,transferred to nitrocellulose membrane, and probed with antibodies tothe indicated proteins. (B and C) Kinase activities of cyclin E, CDK2,and CDK4-associated complexes were assessed during RafAR activation.Lysates (50 μg) prepared as described in (A) were immunoprecipitatedwith the indicated antibodies, and immune-complexes were collected onProtein A-sepharose beads and assayed for kinase activity using histoneH1 (for cyclin E-CDK2) and GST-RB c-terminus (for CDK4) as substrates(B). Quantitation of relative kinase activities in (B) is represented asa percentage of kinase activity from vehicle only (C).

FIG. 3 shows that p21^(Cip1) does not associate with E7 in C4 cells.Lysate was prepared from Babe-transduced cells treated with 1.0 μM R1881for 30 hours. The indicated purified GST fusion proteins were incubatedwith lysate(A) or lysate supplemented with radiolabelled ³⁵S-E7 (B) for12 hours at 4° C. Co-precipitated proteins were separated by 12%SDS-PAGE, transferred to nitrocellulose membrane, and the indicatedproteins detected with the appropriate antibodies (A, both panels, B toppanel) or by phosphorimager analysis (B, bottom panel). Input lanesrepresent 10% of extract or ³⁵S-E7 used per reaction.

FIG. 4 shows E7 does not derepress p21^(Cip1)-associated cyclin E-CDK2.(A) p21^(Cip1) is not associated with active cyclin E-CDK2 in thepresence of E7. Whole cell lysates prepared from RafAR-inducedE7-expressing cells were subjected to three rounds of immunodepletionwith normal rabbit IgG or p21^(Cip1)-specific antibodies. Depletedlysates were analyzed by Western blotting with an antibody specific forp21^(Cip1) (top panel). Immunoprecipitations were performed on depletedlysates with a cyclin E-specific antibody and assayed for histone H1kinase activity (bottom panel) as described in FIG. 2. (B) E7 does notrender cyclin E-CDK2 resistant to p21^(Cip1). Lysates (20 μg) fromasynchronous Babe (▴) or E7 (♦) cells were mixed with increasingconcentrations of purified recombinant GST-p21^(Cip1) and assayed forcyclin E-associated histone H1 kinase activity. Initial activity isrepresented as 100%.

FIG. 5 shows that enhanced cyclin E expression cannot overcomeRafAR-induced arrest. (A) Cyclin E-CDK2 activity was not restored byexogenous cyclin E expression. NIH3T3 cells stably expressing the RafARfusion protein were transduced with constructs expressing empty vector(Babe), HPV-16 E7 (E7), or human cyclin E (Cyclin E). Pools of infectedcells were treated with 0.02% ethanol (−) or 1.0 μM R1881 (+) for 30hours and assessed either for cyclin E-associated (top panels) orCDK2-associated (bottom panels) histone H1 kinase activity. An antibodyspecifically recognizing human cyclin E was utilized forimmunoprecipitating cyclin E-associated kinase activity (top rightpanel) from cells expressing human cyclin E. (B) Exogenous cyclin Eexpression cannot abrogate RafAR-induced arrest. Cell lines were treatedas in (A) and assessed for DNA synthesis via BrdU incorporation.

FIG. 6 shows that E7 alters p21^(Cip1)/cyclin E-CDK2 stoichiometry. (A)Lysates were prepared from Babe- or E7-expressing cells treated with 1.0μM R1881 for 30 hours. The amount of lysate used was standardized tocyclin E steady state levels as determined by Western blotting (toppanel). Cyclin E was immunoprecipitated and immune-complexes wereanalyzed for cyclin E-associated p21^(Cip1) by Western blotting with ap21^(Cip1)-specific antibody (bottom panel). (B) Lysates prepared fromRafAR-induced Babe- or E7-expressing cells were subjected to threerounds of immunodepletion with normal rabbit IgG or p21^(Cip1)-specificantibodies. Depleted lysates were analyzed by Western blotting withantibodies specific for cyclin E (top panel), CDK2 (middle panel), orp21^(Cip1) (bottom panel).

FIG. 7 shows that E7 impairs RafAR-induced p21^(Cip1) nuclearaccumulation. Babe- or E7-expressing cells were treated with 0.02%ethanol (−) or 1.0 μM R1881 for 30 hours, fixed with 3.7%paraformaldehyde, and stained with p21^(Cip1) specific antibody asdescribed in Materials and Methods. Cells were scored for nuclear orwhole-cell localization of p21^(Cip1). (A) Representative fluorescent(left panels) and phase contrast (right panels) micrographs ofRafAR-induced Babe- (upper panels) or E7-expressing cells (lower panels)are shown. (B) Quantitation of cells exhibiting nuclear localization ofp21^(Cip1). The number of cells with nuclear p21^(Cip1) accumulation isrepresented as a percentage of total cells counted. The average anddeviation values shown are from two independent experiments with atleast 200 cells counted per experiment.

FIG. 8 shows that the P13-K/Akt pathway is involved in E7-mediatedabrogation of RafAR-induced arrest. (A) E7-expressing cells were treatedfor 30 hours with 1.0 μM R1881 in the absence or presence of increasingconcentrations of LY294002 (50 μM, 100 μM, 200 μM), an inhibitor ofPI-3K activity. DNA synthesis was measured via BrdU incorporation. (B)E7-expressing cells were treated with 1.0 uM R1881 in the presence orabsence of 100 uM LY294002, stained with p21^(Cip)1-specific antibody,and quantitated for nuclear p21^(Cip1) localization as in FIG. 7. (C)Babe- or E7-expressing cells were co-transfected with a GFP-expressionplasmid (400 ng) and the indicated plasmids (3.2 μg). Aftertransfection, cells were treated with 0.02% ethanol or 1.0 mM R1881 for30 hours, with BrdU added for the last 10 hours of treatment. Cells werestained with α-BrdU, and GFP-positive cells were scored for BrdUincorporation via indirect immunofluorescence. Percentages of BrdUincorporation were calculated by defining the number obtained fromvehicle-treated cells as 100%. The average and deviation values shownare from three independent experiments with at least 150 GFP-positivecells counted per experiment. Low deviation values for some samplescould not be resolved in this figure. (D) Lysates were prepared fromBabe- or E7-expressing cells treated with 0.02% ethanol (−) or 1.0 μMR1881 (+) for 30 hours and analyzed by Western blotting with antibodiesdirected against total Akt (top panel) or Akt phosphorylated on serine473 (P-Akt), representing the active form of Akt. (E) Lysates preparedfrom Babe- or E7-expressing cells treated as in (D) were subjected toimmunoprecipitation with an antibody specific for p21^(Cip1).Precipitated p21^(Cip1) was analyzed for threonine-phosphorylation byWestern blotting with a phospho-threonine-specific antibody (top panel).p21^(Cip1)-expression was analyzed in lysates used for aboveimmunoprecipitations by Western blotting with a p21^(Cip1)-specificantibody (bottom panel).

FIG. 9 shows that the LXCXE motif of E7 is necessary to preventp21^(Cip1)-mediated inhibition of cyclin E-CDK2. (A) NIH3T3 cells stablyexpressing the RafAR fusion protein were transduced with constructsexpressing empty vector (Babe), E7, or E7.C24G. Lysates were preparedand examined for E7 expression with a monoclonal antibody directedagainst HPV-16 E7. (B) Pools of infected cells were treated with 0.02%ethanol (−) or 1.0 μM R1881 (+) for 30 hours and assessed for DNAsynthesis via BrdU incorporation. Low deviation values for some samplescould not be resolved in this figure. (C) Babe-, E7-, orE7.C24G-expressing cells were treated as in (A) and assessed for cyclinE-associated histone H1 kinase activity (top panel) or cellular levelsof active Akt by Western blotting with an antibody specificallyrecognizing Akt phosphorylated on serine 473 (P-Akt, bottom panel).

FIG. 10 shows inhibition of P13-K activity restores p21^(Cip1) nuclearlocalization in E7-expressing cells. E7-expressing cells were treatedfor 30 hours with 1.0 μM R1881 in the absence or presence of 100 μMLY294002, an inhibitor of PI-3K activity. Cells were fixed with 3.7%paraformaldehyde, and stained with p21^(Cip1)-specific antibody asdescribed in Materials and Methods. Cells were scored for nuclear orwhole-cell localization of p21^(Cip1). The number of cells with nuclearp121^(Cip1) accumulation is represented as a percentage of total cellscounted. The average and deviation values shown are from two independentexperiments with at least 200 cells counted per experiment.

FIG. 11 shows p21^(Cip1)-associated cyclin E-CDK2 is inactive inE7-expressing human keratinocytes. Whole cell lysates prepared fromE7-expressing human keratinocytes were subjected to three rounds ofimmunodepletion with normal rabbit IgG or p21^(Cip1)-specificantibodies. Depleted lysates were analyzed by Western blotting with anantibody specific for p21^(Cip1) (top panel). Immunoprecipitations wereperformed on depleted lysates with cyclin E-specific (middle panel) orCDK2-specific (bottom panel) antibodies and assayed for histone H1kinase activity.

FIG. 12 shows that E7 Reduces p27^(Kip1) nuclear accumulation. (A)Control or E7-expressing NIH3T3 cells were grown to confluence andsubsequently subjected to serum-starvation (0.5% BCS) for 24 hours.Cells were collected and analyzed by Western blot withp27^(Kip1)-specific antibody. (B) Confluent, serum-starved cells werefixed with 3.7% paraformaldehyde, and stained with p27^(Kip1)-specificantibody as described in Materials and Methods. Images shown are 40×magnification.

FIG. 13 shows a blot for active Akt (P-Akt) and total Akt (Akt) aftertreatment of NIH3T3 cells with R1881.

FIG. 14 shows that Raf inactivates active Akt by causing thedephosphorylation of Akt at the earelier time points. The drugs andR1881 were added at 0 hr and the samples were taken at 6 hrspost-treatment.

FIG. 15 shows that E7 diminishes TGF-β-induced p27^(Kip1) nuclearlocalization. (A) Tet-E7 Mv1Lu cells were treated for 24 hours with 2μg/mL doxycycline or vehicle. Cell lysates were analyzed by Westernblotting with antibody specific for HPV16 E7. (B) Tet-E7 Mv1Lu cellswere treated for 24 hours with 3 ng/mL TGF-β in the presence or absenceof 2 μg/mL doxycycline and subsequently analyzed for p27^(Kip1)expression via Western blot. (C, D) Tet-E7 Mv1Lu cells were treated asin (B), fixed with 3.7% paraformaldehyde, and stained with p27^(Kip1)specific antibody as described in Materials and Methods. (C)Representative fluorescent (left panels) and phase contrast (rightpanels) micrographs of TGF-β treated cells in the absence (upper panels)or presence (lower panels) of doxycycline. (D) Quantitation of cellsexhibiting nuclear localization of p27^(Kip1). The number of cells withnuclear p27^(Kip1) accumulation is represented as a percentage of totalcells counted. The average and deviation values shown are from twoindependent experiments with at least 200 cells counted per experiment.

FIG. 16 A shows a plot of the % BrdU incorporation as a function ofadriamycin addition in control cells and E7 cells. FIG. 16B shows the %incorporated of P21Cip1 in control and E7 cells. FIG. 16 shows thepresence of the active form of Akt in the presence or absence ofadriamycin and in control cells or E7 cells.

DETAILED DESCRIPTION

The present invention may be understood more readily by reference to thefollowing detailed description of preferred embodiments of the inventionand the Examples included therein and to the Figures and their previousand following description.

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that thisinvention is not limited to specific synthetic methods, specificrecombinant biotechnology methods unless otherwise specified, or toparticular reagents unless otherwise specified, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only and is notintended to be limiting.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint: It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data is provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains. Thereferences disclosed are also individually and specifically incorporatedby reference herein for the material contained in them that is discussedin the sentence in which the reference is relied upon.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

A. COMPOSITIONS AND METHODS

Disclosed herein E7 promotes oncogenesis through an inhibition ofp21^(Cip1) transport into the nucleus. p21^(Cip1) acts as a cell cycleregulator after transport into the nucleus by binding CDK2/cyclincomplexes, such that the cell cycle progression signals of theCDK2/cyclin complex are blocked. Akt is a kinase, which phosphorylatesp21^(Cip1) in the nuclear localization signal (NLS) of p21^(Cip1). Uponphosphorylation nuclear transport does not occur and p21^(Cip1) isubiquinated and subsequently degraded. Disclosed herein, Raf inducescell cycle arrest through increasing the level of p21^(Cip1) in a celland in the nucleus. Disclosed herein, Raf accomplishes this byincreasing the expression of p21^(l Cip1) as well as promoting thedegradation of Akt, the negative regulator of p21^(Cip1). The HPVprotein, E7 abbrogates the G1 cell cycle arrest. Disclosed herein, E7accomplishes this abrogation not by decreasing the expression ofp21^(Cip1), but rather by preventing the degradation of Akt. Thus, E7 isan Akt activity promoting molecule. Furthermore, E7 is an Aktmaintaining molecule. This effect of E7 on Akt allows for theidentifification and production of molecules that inhibit E7 oncogeniceffects on a cell.

The cellular response to oncogenic Ras depends upon the presence orabsence of cooperating mutations. In the absence of immortalizingoncogenes or genetic lesions, activation of the Ras/Raf pathway resultsin a p21^(Cip1)-dependent cellular arrest. The human papillomavirusoncoprotein E7 transforms primary cells in cooperation with Ras andabrogates p21^(Cip1)-mediated growth arrest in the presence of variousantimitogenic signals. Disclosed is a conditional Raf molecule toinvestigate the effects of E7 on p21^(Cip1) function in the context ofRaf-induced cellular arrest. E7 bypassed Raf-induced arrest andalleviated inhibition of cyclin E-CDK2 without suppressing Raf-specificsynthesis of p21^(Cip1) or derepressing p21^(Cip1)-associated CDK2complexes. Activation of Raf led to nuclear accumulation of p21^(Cip1),and provided herein is evidence that this effect is mediated byinhibition of Akt, a regulator of p21^(Cip1) localization. Disclosedherein, loss of Akt activity is an important event in the cellulararrest associated with Raf-induction, since maintenance of Akt activitywas necessary and sufficient to bypass Raf-induced arrest. In agreement,expression of E7 sustained Akt activity and reduced nuclear accumulationof p21^(Cip1), resulting in decreased association between p21^(Cip1) andcyclin E-CDK2. Disclosed herein, E7 inhibits p21^(Cip1) function in thecontext of Raf signaling by altering Raf-Akt antagonism and preventingthe proper subcellular localization of p21^(Cip1).

Disclosed herein E7 can maintain Akt activity when cells are given anarrest signal, such as that caused by cRaf-1, and that this results inthe cells being able to bypass the arrest. One of the arrest signalsused was the over-expression of cRaf-1. Disclosed herein cRaf-1 caninactivate Akt. Also disclosed E7 can abrogate this inhibition and somaintain Akt activity. Disclosed herein the ability of HPV-16 E7 tobypass the arrest is dependent on the capacity of E7 to bind andinactivate Rb. This is consistent with E7 affecting the transcriptionalregulation of a gene(s) that control Akt activity.

Disclosed herein the inactivation of Akt occurs within 3 to 6 hoursafter induction of cRaf-1. Cell cycle arrest is not evident until 16 to20 hours after induction of cRaf-1. Also disclosed, the Akt proteinslevels do not change at the early time points, but the active,phosphorylated form of Akt disappears at the early time points. It isalso shown herein that cRaf-1 inhibits Akt activity through MEK1(MAPkinasekinase1), which is a downstream target of cRaf-1, using MEK1inhibitors. Furthermore, new protein synthesis is required for theinhibition of Akt, by the cell cycle arrestor. This was shown usingcyclohexamide, which interferes with protein translation. It is alsoshown herein that degradation of activate Akt through the proteosomepathway does not occur, and this was shown through using proteosomeinhibitors. Also disclosed herein the upstream effector of Akt, namelyPI3 kinase activity, is not affected by the cRaf-1. Overall theseresults indicate that the inactivation of Akt is through a phosphataseenzyme that removes the activating phosphate group on Akt. The data isconsistent with E7 controlling the activity of the phosphatase.

Systems designed to enhance this E7 effect are disclosed and thesesystems can be used for identifying molecules that modulate this effect.Thus, disclosed are compositions and methods related to identifyingmolecules that affect E7 related events in cells. Also disclosed arecompoisitions that inhibit the E7 maintenance of Akt activity. Alsodisclosed herein E7 does not need to alter the amount of Akt geneexpression to modulate the amount of Akt in the cell.

B. COMPOSITIONS

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular E7 is disclosed and discussed and a number ofmodifications that can be made to a number of molecules including the E7are discussed, specifically contemplated is each and every combinationand permutation of E7 and the modifications that are possible unlessspecifically indicated to the contrary. Thus, if a class of molecules A,B, and C are disclosed as well as a class of molecules D, E, and F andan example of a combination molecule, A-D is disclosed, then even ifeach is not individually recited each is individually and collectivelycontemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E,and C-F are considered disclosed. Likewise, any subset or combination ofthese is also disclosed. Thus, for example, the sub-group of A-E, B-F,and C-E would be considered disclosed. This concept applies to allaspects of this application including, but not limited to, steps inmethods of making and using the disclosed compositions. Thus, if thereare a variety of additional steps that can be performed it is understoodthat each of these additional steps can be performed with any specificembodiment or combination of embodiments of the disclosed methods.

The disclosed compositions and methods are involved in the modulation ofthe cell cycle. The disclosed compositions and methods utilize theinformation disclosed herein that E7 regulates p21^(Cip1) activity bypreventing p21^(Cip1) import into the nucleus through E7 regulation ofAkt, a regulator of p21^(Cip1). Disclosed herein, E7 is shown toregulate Akt by down regulating Akt degradation.

1. Molecules Involved in the Cell Cycle

Regulation of mammalian cell proliferation is governed by signalingpathways that ultimately converge on the activity of cyclin-dependentkinases (CDKs). Progression through the G1 phase of the mitotic cellcycle is controlled by the activities of the G1 CDKs, cyclin D-CDK4/6and cyclin E-CDK2 (Sherr, 1994a; Sherr, 1996). Cyclins D and E arerate-limiting for progression through G1, as both cyclins shorten G1when overexpressed and are essential for G1-S transition in highereukaryotes (Ohtsubo et al., 1995; Quelle et al., 1993). An importantfunction of the G1 CDKs is to phosphorylate and inactivate theretinoblastoma tumor suppressor protein (RB) (Sherr, 1994b; Weinberg,1995). RB is a negative regulator of cell proliferation that exerts itseffects, at least in part, by associating with various transcriptionfactors (in particular, members of the E2F/DP family) and repressingtranscription (Weinberg, 1995). The targets of RB-mediated regulationinclude genes required for G1-S progression (E2F1, cyclin E, cyclin A)and DNA synthesis (DNA polα, DHFR, thymidine synthetase), underliningthe central role of RB in governing cell proliferation (Weinberg, 1995).In addition, cyclin E- and A-CDK2 complexes have essential non-RBsubstrates, as attenuation of CDK2 arrests cells in G1 in the absence ofa functional RB pathway (Alevizopoulos et al., 1997; Hofmann andLivingston, 1996; Lukas et al., 1997).

a) Cyclin Dependent Kinase Inhibitors

The activity of CDKs is regulated at multiple tiers includingassociation with cyclins, nuclear localization, activating andinactivating phosphorylations, and association with specificCDK-inhibitors (CKIs) (Morgan, 1995). The physiologic importance of CKIsis emphasized by the observation that many antiproliferative signalslead to increased expression of these inhibitory molecules and thatcells deficient for expression of specific CKIs demonstrate defects incell cycle control (Harper and Elledge, 1996; Sherr and Roberts, 1995).Mammalian CKIs are divided into two classes based on primary sequencehomology. The INK4 family of CKIs (p16^(Ink4a), p15^(Ink4b),p18^(Ink4c), p19^(Ink4d)) interacts with and inhibits CDK4/6 bypreventing their association with D-type cyclins. CKIs of the Kip/Cipfamily (p21^(Cip1), p27^(Kip1), p57^(Kip2)) inhibit a broader spectrumof CDKs.

b) p21^(Cip1)

P21^(Cip1) associates with complexes of cyclins D1/D2/D3-CDK4/6 andcyclins E/A-CDK2 (Harper et al., 1993; Harper et al., 1995). TheN-terminus of p21^(Cip1) is conserved among Kip/Cip CKIs and containscyclin- and CDK-binding sites that are necessary for inhibiting CDKactivity and cell cycle arrest (Sherr and Roberts, 1995). The C-terminalhalf of p21^(Cip1) contains a second cyclin-binding motif, aPCNA-binding domain, and a nuclear localization sequence (NLS).p21^(Cip1) is a potent inhibitor of CDK2 in vivo. Indeed, CDK2represents an important target of p21^(Cip1) in regulatingproliferation, since numerous antimitogenic stimuli, including growthfactor starvation, contact-inhibition, anchorage detachment, DNA damage,and TGF-β, result in Kip/Cip-mediated inhibition of cyclin E-CDK2 andsubsequent cell cycle arrest (Coats et al., 1996; Dulic et al., 1994;Fang et al., 1996; Koff et al., 1993; Polyak et al., 1994a; Polyak etal., 1994b). Transcription of p21^(Cip1) is activated by the p53 tumorsuppressor in response to DNA damage, and p21^(Cip1) is an essentialdeterminant of p53-induced G1 arrest (el-Deiry et al., 1993). Inagreement, p21Cip1−/− embyronic fibroblasts do not undergo G1 arrestfollowing genotoxic stress or nucleotide perturbation (Brugarolas etal., 1995; Deng et al., 1995). p21^(Cip1) has also been implicated inp53-independent senescence-derived arrest and in terminaldifferentiation of myoblast, epithelial, and hematopoietic cell lineages(Missero et al., 1995; Missero et al., 1996; Noda et al., 1994; Parkeret al., 1995). In addition, it has been suggested that p21^(Cip1) is animportant target in the oncogenic activity of HER-2/neu and Akt, sincephosphorylation of the p21^(Cip1) NLS by Akt, a downstream effector ofthe PI3-K signaling pathway, has recently been shown to alter thesubcellular localization and cell-growth inhibitory function ofp21^(Cip1) (Zhou et al., 2001).

2. Human Papillomaviruses (HPV)

Human papillomaviruses (HPV) are small DNA viruses that requireunscheduled S phase entry in terminally differentiated epithelialkeratinocytes in order for viral genome amplification to occur (Laimins,1993). Not surprisingly, HPV have evolved several strategies foruncoupling differentiation from cell cycle arrest. The E7 early geneproduct of HPV16 stimulates cellular progression through the G I -Stransition in the presence of various G1-arrest signals and canimmortalize and transform several cell types alone or in cooperationwith activated ras (Banks et al., 1990; Edmonds and Vousden, 1989;Phelps et al., 1992), suggesting E7 has evolved to interact with keycomponents of cellular growth-regulatory pathways.

E7 has been shown to target the RB family of pocket proteins (RB, p 107,p 130) (Halpern, 1997). E7 interacts with the pocket proteins through anLXCXE motif and can disrupt RB-mediated gene regulation. In addition,expression of E7 leads to a reduction in the steady state level of RB byubiquitin-dependent degradation (Boyer et al., 1996). E7 also abrogatesG1 arrest induced by DNA damage, epithelial differentiation, TGF-β,growth factor withdrawal, and anchorage detachment (Banks et al., 1990;Demers et al., 1996; Funk et al., 1997; Hickman et al., 1997; Jones andMunger, 1997; Ruesch and Laimins, 1998; Schulze et al., 1998), stimulithat negatively regulate proliferation via p21^(Cip1) or the closelyrelated p27^(Kip1).

Premalignant lesions caused by HPV occur in all stratified squamousepithelia and E7 is essential for the generation of new infectious virusand the abnormalities observed in the infected epithelium. E7 inducesAKT activity, which over-rides the inhibitory activity of p21CIP. Thereare other cancers, such as breast cancer, where there is an increase inthe kinase inhibitor, p27KIP (related to p21CIP). There is also arelationship between the high level of p27KIP and poor prognosis for thepatient. The cellular localization of the p27KIP in these cancers issimilar to that of E7-expressing cells and suggests that there is acelluar function, which is altered in these breast cancer cells which isalso altered in E7-expressing cells.

3. Systems Which can be Used to Identify Modulators of E7 Activity

Disclosed are systems which can be used to identify compounds thataffect the E7 maintenance of Akt in a cell. There a number of componentswhich are present in these systems. It is understandable that thecomponents are general and that they may be substituted with functionalequivalents. One aspect of the systems is that the systems should beable to up-regulate p21^(Cip1) production. One way of doing this is tohave a regulatable Raf gene present in the system, expressed from, forexample, a vector encoding a Raf gene under the control of a regulatablepromoter. A system incorporating Raf in this manner allows forcontollable upregulation of p21^(Cip1) production in the system in aphysiological manner.

The systems, also will typically express Akt, or some form of Akt. It isunderstood that there are active forms of Akt (phosphorylated) and inactive forms of Akt (unphosphorylated).

The system also typically will include a means of expressing E7, such asvector encoding E7. This aspect of the system allows for the E7maintenance of Akt to be observed. The expression of E7 can either beregulatable or constitutive.

The system then can comprise a variety of components, such as potentialinihibtors of E7 or PI3K or active Akt. The system could, for example,comprise a dominant-negative mutant of Akt, such as Akt K179M. This typeof system can be used as a control, for example, to compare theinihibtor effects of potential Akt inihibitors, by for example,comparing the potential inhibitors activity to that of the inhibitor AktK179M, which is disclosed herein. The system can also further include ameans for providing E7 point mutants, such as vectors expressing E7point mutations. For example, the E7 point mutation may be the E7.C24Gpoint mutation, which is a mutation that prevents interaction with Rb.The point mutation has reduced ability to bind Akt. This type of systemcan also be used as a control, for comparing the effect of otherinhibitors of Akt maintenance activity. Typically, only E7 is required.Also, typically only the enzymatic domain of Raf is required along witha localization signal for obtaining appropriate Raf function within thesystems. [Sewing et al. 1997 Mol. Cell Biol. 17: 5588-5597].

Also disclosed are systems which have a regulatable Raf component aswell as a component that produces stable expression of human cyclin E.

All of the various components can be expressed in a variety of ways. Forexample, they can be constitutively expressed as described herein, orregulatable. Furthermore, they can be expressed through activation ofendogenous genes, for example in cell types which may not normallyexpress one or more of the proteins, but which may have a beneficialbackground for looking at the effects of E7. It is also understoodherein that functional variants and alleles exist for all of theproteins making up the disclosed systems.

These systems can be utilized in a variety of cells, such as NIH3T3.Other cell types such as human foreskin keratinocytes (HFK) can be usedwhen arrested through DNA damage. Also NIH3T3 cells arrested by serumstarvation can be used. In this latter case p27^(KIP) is the cyclinkinase inhibitor which is bypassed. One of the cell types that can beused is epithelial cells. For example mink lung cells, such as Mv1Lu,can be used. Typically the cells used must be capable of arrest using apathway that involves Akt. For example any type of cell, immortalizedwith Raf or Raf component, such as cRaf-1, will have these properties.

The systems will typically contain or be under some type of arrestsignal. For example, the cells can be arrested by Raf or a Raf variant,or cRaf-1. The cells can also be arrested through for example, DNAdamage, such as that caused by Adriamycin (see Example 3). The cells canalso be arrested by TGF-β or by serum starvation. Cells can also bearrested by differentiation, for example human keratinocytes (HFK).

The systems are designed such that various activities or components orstates, for example, can be assayed. These compnonents, activities, orstates, for example, are related as disclosed herein to the abrogationby E7 of the arrest of the cell cycle through the effects on Akt. Forexample, the amount of active Akt or total Akt, can be measured in thesystems as an indication of the effect E7 is having on the system, aswhen cells are arrested as disclosed herein, there is ultimate decreasein active Akt amount. Furthermore, the amount of active Akt,phosphprylated, can be measured, by for example, probing with antibodiesto the phosphorylated form. Furthermore, the activity of Akt can bemeasured, by for example, looking at P21^(cip) presence. The systems canalso look at a variety of factors, such as MEK-1 activity or P21Cipactivity, or cyclin accumulation or CDK-cyclin complex formation or Rbactivity, or E7-Rb complex formation, which are related to the arrest ofcells and the abrogation of this arrest by E7.

It is understood that the disclosed methods and systems for identifyingmolecules that inhibit E7 abbrogation, for example, can be performedusing high through put means. For example, putative inhibitors can beidentified using Fluorescence Resonance Energy Transfer (FRET) toquickly identify interactions. The underlying theory of the techniquesis that when two molecules are close in space, ie, interacting at alevel beyond background, a signal is produced or a signal can bequenched. Then, a variety of experiments can be performed, including,for example, adding in a putative inhibitor. If the inhibitor competeswith the interaction between the two signaling molecules, the signalswill be removed from each other in space, and this will cause a decreaseor an increase in the signal, depending on the type of signal used. Thisdecreasing or increasing signal can be correlated to the presence orabsence of the putative inhibitor. Any signaling means can be used. Forexample, disclosed are methods of identifying an inhibitor of theinteraction between any two of the disclosed molecules comprising,contacting a first molecule and a second molecule together in thepresence of a putative inhibitor, wherein the first molecule or secondmolecule comprises a fluorescence donor, wherein the first or secondmolecule, typically the molecule not comprising the donor, comprises afluorescence acceptor; and measuring Fluorescence Resonance EnergyTransfer

In general, it is understood that one way to define any known variantsand derivatives or those that might arise, of the disclosed genes andproteins herein, is through defining the variants and derivatives interms of homology to specific known sequences. This identity ofparticular sequences disclosed herein is also discussed elsewhereherein. In general, variants of genes and proteins herein disclosedtypically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 99 percent homology to the stated sequence or the nativesequence. Those of skill in the art readily understand how to determinethe homology of two proteins or nucleic acids, such as genes. Forexample, the homology can be calculated after aligning the two sequencesso that the homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment. It isunderstood that any of the methods typically can be used and that incertain instances the results of these various methods may differ, butthe skilled artisan understands if identity is found with at least oneof these methods, the sequences would be said to have the statedidentity, and be disclosed herein.

For example, as used herein, a sequence recited as having a particularpercent homology to another sequence refers to sequences that have therecited homology as calculated by any one or more of the calculationmethods described above. For example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingthe Zuker calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by any of theother calculation methods. As another example, a first sequence has 80percent homology, as defined herein, to a second sequence if the firstsequence is calculated to have 80 percent homology to the secondsequence using both the Zuker calculation method and the Pearson andLipman calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by the Smith andWaterman calculation method, the Needleman and Wunsch calculationmethod, the Jaeger calculation methods, or any of the other calculationmethods. As yet another example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingeach of calculation methods (although, in practice, the differentcalculation methods will often result in different calculated homologypercentages).

5. Hybridization/Selective Hybridization

The term hybridization typically means a sequence driven interactionbetween at least two nucleic acid molecules, such as a primer or a probeand a gene. Sequence driven interaction means an interaction that occursbetween two nucleotides or nucleotide analogs or nucleotide derivativesin a nucleotide specific manner. For example, G interacting with C or Ainteracting with T are sequence driven interactions. Typically sequencedriven interactions occur on the Watson-Crick face or Hoogsteen face ofthe nucleotide. The hybridization of two nucleic acids is affected by anumber of conditions and parameters known to those of skill in the art.For example, the salt concentrations, pH, and temperature of thereaction all affect whether two nucleic acid molecules will hybridize.

Parameters for selective hybridization between two nucleic acidmolecules are well known to those of skill in the art. For example, insome embodiments selective hybridization conditions can be defined asstringent hybridization conditions. For example, stringency ofhybridization is controlled by both temperature and salt concentrationof either or both of the hybridization and washing steps. For example,the conditions of hybridization to achieve selective hybridization mayinvolve hybridization in high ionic strength solution (6×SSC or 6×SSPE)at a temperature that is about 12-25° C. below the Tm (the meltingtemperature at which half of the molecules dissociate from theirhybridization partners) followed by washing at a combination oftemperature and salt concentration chosen so that the washingtemperature is about 5° C. to 20° C. below the Tm. The temperature andsalt conditions are readily determined empirically in preliminaryexperiments in which samples of reference DNA immobilized on filters arehybridized to a labeled nucleic acid of interest and then washed underconditions of different stringencies. Hybridization temperatures aretypically higher for DNA-RNA and RNA-RNA hybridizations. The conditionscan be used as described above to achieve stringency, or as is known inthe art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989;Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is hereinincorporated by reference for material at least related to hybridizationof nucleic acids). A preferable stringent hybridization condition for aDNA:DNA hybridization can be at about 68° C. (in aqueous solution) in6×SSC or 6×SSPE followed by washing at 68° C. Stringency ofhybridization and washing, if desired, can be reduced accordingly as thedegree of complementarity desired is decreased, and further, dependingupon the G-C or A-T richness of any area wherein variability is searchedfor. Likewise, stringency of hybridization and washing, if desired, canbe increased accordingly as homology desired is increased, and further,depending upon the G-C or A-T richness of any area wherein high homologyis desired, all as known in the art.

Another way to define selective hybridization is by looking at theamount (percentage) of one of the nucleic acids bound to the othernucleic acid. For example, in some embodiments selective hybridizationconditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid isbound to the non-limiting nucleic acid. Typically, the non-limitingprimer is in for example, 10 or 100 or 1000 fold excess. This type ofassay can be performed at under conditions where both the limiting andnon-limiting primer are for example, 10 fold or 100 fold or 1000 foldbelow their k_(d), or where only one of the nucleic acid molecules is 10fold or 100 fold or 1000 fold or where one or both nucleic acidmolecules are above their k_(d).

Another way to define selective hybridization is by looking at thepercentage of primer that gets enzymatically manipulated underconditions where hybridization is required to promote the desiredenzymatic manipulation. For example, in some embodiments selectivehybridization conditions would be when at least about, 60, 65, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer isenzymatically manipulated under conditions which promote the enzymaticmanipulation, for example if the enzymatic manipulation is DNAextension, then selective hybridization conditions would be when atleast about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100percent of the primer molecules are extended. Preferred conditions alsoinclude those suggested by the manufacturer or indicated in the art asbeing appropriate for the enzyme performing the manipulation.

Just as with homology, it is understood that there are a variety ofmethods herein disclosed for determining the level of hybridizationbetween two nucleic acid molecules. It is understood that these methodsand conditions may provide different percentages of hybridizationbetween two nucleic acid molecules, but unless otherwise indicatedmeeting the parameters of any of the methods would be sufficient. Forexample if 80% hybridization was required and as long as hybridizationoccurs within the required parameters in any one of these methods it isconsidered disclosed herein.

It is understood that those of skill in the art understand that if acomposition or method meets any one of these criteria for determininghybridization either collectively or singly it is a composition ormethod that is disclosed herein.

6. Nucleic Acids

There are a variety of molecules disclosed herein that are nucleic acidbased, including for example the nucleic acids that encode, for example,as well as various functional nucleic acids. The disclosed nucleic acidsare made up of for example, nucleotides, nucleotide analogs, ornucleotide substitutes. Non-limiting examples of these and othermolecules are discussed herein. It is understood that for example, whena vector is expressed in a cell, that the expressed mRNA will typicallybe made up of A, C, G, and U. Likewise, it is understood that if, forexample, an antisense molecule is introduced into a cell or cellenvironment through for example exogenous delivery, it is advantagousthat the antisense molecule be made up of nucleotide analogs that reducethe degradation of the antisense molecule in the cellular environment.

a) Nucleotides and Related Molecules

A nucleotide is a molecule that contains a base moiety, a sugar moietyand a phosphate moiety. Nucleotides can be linked together through theirphosphate moieties and sugar moieties creating an internucleosidelinkage. The base moiety of a nucleotide can be adenin-9-yl (A),cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).The sugar moiety of a nucleotide is a ribose or a deoxyribose. Thephosphate moiety of a nucleotide is pentavalent phosphate. Anon-limiting example of a nucleotide would be 3′-AMP (3′-adenosinemonophosphate) or 5′-GMP (5′-guanosine monophosphate).

A nucleotide analog is a nucleotide which contains some type ofmodification to either the base, sugar, or phosphate moieties.Modifications to nucleotides are well known in the art and would includefor example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, and 2-aminoadenine as well as modifications atthe sugar or phosphate moieties.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid.

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989,86, 6553-6556),

A Watson-Crick interaction is at least one interaction with theWatson-Crick face of a nucleotide, nucleotide analog, or nucleotidesubstitute. The Watson-Crick face of a nucleotide, nucleotide analog, ornucleotide substitute includes the C2, N1, and C6 positions of a purinebased nucleotide, nucleotide analog, or nucleotide substitute and theC2, N3, C4 positions of a pyrimidine based nucleotide, nucleotideanalog, or nucleotide substitute.

A Hoogsteen interaction is the interaction that takes place on theHoogsteen face of a nucleotide or nucleotide analog, which is exposed inthe major groove of duplex DNA. The Hoogsteen face includes the N7position and reactive groups (NH2 or O) at the C6 position of purinenucleotides.

b) Sequences

There are a variety of sequences related to, for example, the E7 gene,the Akt gene, the p21^(Cip1) gene, and the Raf gene, and there encodedproducts, are set forth in SEQ ID Nos: 1-13. Also disclosed arefragments, functional fragments, and individual subsequences containedwithin these sequences. It is understood that there are a variety ofallelic sequences as well as strain variants of the disclosed proteinsand the nucleic acids that encode them. For example, oncogenic humanpapilloma virus (HPVs) contain a varity of strain variants sequencesencoding for E7, but all of these proteins have a similar functionalcapability.

One particular sequence set forth in SEQ ID NO: 9, encoding E7 is usedherein to exemplify the disclosed compositions and methods. It isunderstood that the description related to this sequence is applicableto any sequence related to SEQ ID NO:9 or the other disclosed sequences,unless specifically indicated otherwise. Those of skill in the artunderstand how to resolve sequence discrepancies and differences and toadjust the compositions and methods relating to a particular sequence toother related sequences (i.e. sequences of E7, Akt, p21^(Cip1), or Raf).Primers and/or probes can be designed for any E7, Akt, p21^(Cip1), orRaf sequence given the information disclosed herein and known in theart.

c) Primers and Probes

Disclosed are compositions including primers and probes, which arecapable of interacting with the disclosed nucleic acids including thosefor p21^(Cip1), Akt, E7, and Raf, as disclosed herein. In certainembodiments the primers are used to support DNA amplification reactions.Typically the primers will be capable of being extended in a sequencespecific manner. Extension of a primer in a sequence specific mannerincludes any methods wherein the sequence and/or composition of thenucleic acid molecule to which the primer is hybridized or otherwiseassociated directs or influences the composition or sequence of theproduct produced by the extension of the primer. Extension of the primerin a sequence specific manner therefore includes, but is not limited to,PCR, DNA sequencing, DNA extension, DNA polymerization, RNAtranscription, or reverse transcription. Techniques and conditions thatamplify the primer in a sequence specific manner are preferred. Incertain embodiments the primers are used for the DNA amplificationreactions, such as PCR or direct sequencing. It is understood that incertain embodiments the primers can also be extended using non-enzymatictechniques, where for example, the nucleotides or oligonucleotides usedto extend the primer are modified such that they will chemically reactto extend the primer in a sequence specific manner. Typically thedisclosed primers hybridize with the p21^(Cip1), Akt, E7, and Raf genes,for example, or region of the p21^(Cip1), Akt, E7, and Raf genes or theyhybridize with the complement of the p21^(Cip1), Akt, E7, and Raf genes,for example, or complement of a region of the p21^(Cip1), Akt, E7, andRaf genes.

d) Functional Nucleic Acids

Functional nucleic acids are nucleic acid molecules that have a specificfunction, such as binding a target molecule or catalyzing a specificreaction. Functional nucleic acid molecules can be divided into thefollowing categories, which are not meant to be limiting. For example,functional nucleic acids include antisense molecules, aptamers,ribozymes, triplex forming molecules, and external guide sequences. Thefunctional nucleic acid molecules can act as affectors, inhibitors,modulators, and stimulators of a specific activity possessed by a targetmolecule, or the functional nucleic acid molecules can possess a de novoactivity independent of any other molecules.

Functional nucleic acid molecules can interact with any macromolecule,such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functionalnucleic acids can interact with the mRNA of, for example, E7 or Akt orthe genomic DNA of E7 or Akt or they can interact with the E7 or Aktpolypeptides. Often functional nucleic acids are designed to interactwith other nucleic acids based on sequence homology between the targetmolecule and the functional nucleic acid molecule. In other situations,the specific recognition between the functional nucleic acid moleculeand the target molecule is not based on sequence homology between thefunctional nucleic acid molecule and the target molecule, but rather isbased on the formation of tertiary structure that allows specificrecognition to take place.

Antisense molecules are designed to interact with a target nucleic acidmolecule through either canonical or non-canonical base pairing. Theinteraction of the antisense molecule and the target molecule isdesigned to promote the destruction of the target molecule through, forexample, RNAseH mediated RNA-DNA hybrid degradation. Alternatively theantisense molecule is designed to interrupt a processing function thatnormally would take place on the target molecule, such as transcriptionor replication. Antisense molecules can be designed based on thesequence of the target molecule. Numerous methods for optimization ofantisense efficiency by finding the most accessible regions of thetarget molecule exist. Exemplary methods would be in vitro selectionexperiments and DNA modification studies using DMS and DEPC. It ispreferred that antisense molecules bind the target molecule with adissociation constant (k_(d))less than 10⁻⁶. It is more preferred thatantisense molecules bind with a k_(d) less than 10⁻⁸. It is also morepreferred that the antisense molecules bind the target moelcule with ak_(d) less than 10⁻¹⁰. It is also preferred that the antisense moleculesbind the target molecule with a k_(d) less than 10⁻¹². A representativesample of methods and techniques which aid in the design and use ofantisense molecules can be found in the following non-limiting list ofU.S. Pat. Nos. 5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317,5,780,607, 5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590,5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522,6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004,6,046,319, and 6,057,437.

Aptamers are molecules that interact with a target molecule, preferablyin a specific way. Typically aptamers are small nucleic acids rangingfrom 15-50 bases in length that fold into defined secondary and tertiarystructures, such as stem-loops or G-quartets. Aptamers can bind smallmolecules, such as ATP (U.S. Pat. No. 5,631,146) and theophiline (U.S.Pat. No. 5,580,737), as well as large molecules, such as reversetranscriptase (U.S. Pat. No. 5,786,462) and thrombin (U.S. Pat. No.5,543,293). Aptamers can bind very tightly with kds from the targetmolecule of less than 10⁻¹² M. It is preferred that the aptamers bindthe target molecule with a k_(d) less than 10⁻⁶. It is more preferredthat the aptamers bind the target molecule with a k_(d) less than 10⁻⁸.It is also more preferred that the aptamers bind the target moleculewith a k_(d) less than 10⁻¹⁰. It is also preferred that the aptamersbind the target molecule with a k_(d) less than 10⁻¹². Aptamers can bindthe target molecule with a very high degree of specificity. For example,aptamers have been isolated that have greater than a 10000 folddifference in binding affinities between the target molecule and anothermolecule that differ at only a single position on the molecule (U.S.Pat. No. 5,543,293). It is preferred that the aptamer have a k_(d) withthe target molecule at least 10 fold lower than the k_(d) with abackground binding molecule. It is more preferred that the aptamer havea k_(d) with the target molecule at least 100 fold lower than the k_(d)with a background binding molecule. It is more preferred that theaptamer have a k_(d) with the target molecule at least 1000 fold lowerthan the k_(d) with a background binding molecule. It is preferred thatthe aptamer have a k_(d) with the target molecule at least 10000 foldlower than the k_(d) with a background binding molecule. It is preferredwhen doing the comparison for a polypeptide for example, that thebackground molecule be a different polypeptide. For example, whendetermining the specificity of E7 or Akt aptamers, the backgroundprotein could be serum albumin. Representative examples of how to makeand use aptamers to bind a variety of different target molecules can befound in the following non-limiting list of U.S. Pat. Nos. 5,476,766,5,503,978, 5,631,146, 5,731,424, 5,780,228, 5,792,613, 5,795,721,5,846,713, 5,858,660, 5,861,254, 5,864,026, 5,869,641, 5,958,691,6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776, and6,051,698.

Ribozymes are nucleic acid molecules that are capable of catalyzing achemical reaction, either intramolecularly or intermolecularly.Ribozymes are thus catalytic nucleic acid. It is preferred that theribozymes catalyze intermolecular reactions. There are a number ofdifferent types of ribozymes that catalyze nuclease or nucleic acidpolymerase type reactions which are based on ribozymes found in naturalsystems, such as hammerhead ribozymes, (for example, but not limited tothe following U.S. Pat. Nos. 5,334,711, 5,436,330, 5,616,466, 5,633,133,5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288,5,891,683, 5,891,684,5,985,621, 5,989,908, 5,998,193, 5,998,203, WO9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO9718312 by Ludwig and Sproat) hairpin ribozymes (for example, but notlimited to the following U.S. Pat. Nos. 5,631,115, 5,646,031, 5,683,902,5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), andtetrahymena ribozymes (for example, but not limited to the followingU.S. Pat. Nos. 5,595,873 and 5,652,107). There are also a number ofribozymes that are not found in natural systems, but which have beenengineered to catalyze specific reactions de novo (for example, but notlimited to the following U.S. Pat. Nos. 5,580,967, 5,688,670, 5,807,718,and 5,910,408). Preferred ribozymes cleave RNA or DNA substrates, andmore preferably cleave RNA substrates. Ribozymes typically cleavenucleic acid substrates through recognition and binding of the targetsubstrate with subsequent cleavage. This recognition is often basedmostly on canonical or non-canonical base pair interactions. Thisproperty makes ribozymes particularly good candidates for targetspecific cleavage of nucleic acids because recognition of the targetsubstrate is based on the target substrates sequence. Representativeexamples of how to make and use ribozymes to catalyze a variety ofdifferent reactions can be found in the following non-limiting list ofU.S. Pat. Nos. 5,646,042,5,693,535, 5,731,295, 5,811,300, 5,837,855,5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704, 5,989,906, and6,017,756.

Triplex forming functional nucleic acid molecules are molecules that caninteract with either double-stranded or single-stranded nucleic acid.When triplex molecules interact with a target region, a structure calleda triplex is formed, in which there are three strands of DNA forming acomplex dependant on both Watson-Crick and Hoogsteen base-pairing.Triplex molecules are preferred because they can bind target regionswith high affinity and specificity. It is preferred that the triplexforming molecules bind the target molecule with a k_(d) less than 10⁻⁶.It is more preferred that the triplex forming molecules bind with ak_(d) less than 10⁻⁸. It is also more preferred that the triplex formingmolecules bind the target moelcule with a k_(d) less than 10⁻¹⁰. It isalso preferred that the triplex forming molecules bind the targetmolecule with a k_(d) less than 10⁻¹². Representative examples of how tomake and use triplex forming molecules to bind a variety of differenttarget molecules can be found in the following non-limiting list of U.S.Pat. Nos. 5,176,996, 5,645,985, 5,650,316,5,683,874, 5,693,773,5,834,185, 5,869,246, 5,874,566, and 5,962,426.

External guide sequences (EGSs) are molecules that bind a target nucleicacid molecule forming a complex, and this complex is recognized by RNaseP, which cleaves the target molecule. EGSs can be designed tospecifically target a RNA molecule of choice. RNAse P aids in processingtransfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited tocleave virtually any RNA sequence by using an EGS that causes the targetRNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 byYale, and Forster and Altman, Science 238:407-409 (1990)).

Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can beutilized to cleave desired targets within eukarotic cells. (Yuan et al.,Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO 93/22434 by Yale; WO95/24489 by Yale; Yuan and Altman, EMBO J 14:159-168 (1995), and Carraraet al., Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995)).Representative examples of how to make and use EGS molecules tofacilitate cleavage of a variety of different target molecules be foundin the following non-limiting list of U.S. Pat. Nos. 5,168,053,5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162

7. Antibodies

a) Antibodies Generally

The term “antibodies” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. In addition to intactimmunoglobulin molecules, also included in the term “antibodies” arefragments or polymers of those immunoglobulin molecules, and human orhumanized versions of immunoglobulin molecules or fragments thereof, aslong as they are chosen for their ability to interact with E7 or Aktsuch that the disclosed effect on p21^(Cip1). The antibodies can betested for their desired activity using the in vitro assays describedherein, or by analogous methods, after which their in vivo therapeuticand/or prophylactic activities are tested according to known clinicaltesting methods. Also disclosed are functional equivalents ofantibodies.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies within the population are identicalexcept for possible naturally occurring mutations that may be present ina small subset of the antibody molecules. The monoclonal antibodiesherein specifically include “chimeric” antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, as long as they exhibit the desired antagonisticactivity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl.Acad. Sci. USA, 81:6851-6855 (1984)).

The disclosed monoclonal antibodies can be made using any procedurewhich produces mono clonal antibodies. For example, monoclonalantibodies of the invention can be prepared using hybridoma methods,such as those described by Kohler and Milstein, Nature, 256:495 (1975).In a hybridoma method, a mouse or other appropriate host animal istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNAencoding the disclosed monoclonal antibodies can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). Libraries of antibodies oractive antibody fragments can also be generated and screened using phagedisplay techniques, e.g., as described in U.S. Pat. No. 5,804,440 toBurton et al. and U.S. Pat. No. 6,096,441 to Barbas et al.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fc fragment.Pepsin treatment yields a fragment that has two antigen combining sitesand is still capable of cross-linking antigen.

The fragments, whether attached to other sequences or not, can alsoinclude insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the antibody or antibody fragment is notsignificantly altered or impaired compared to the non-modified antibodyor antibody fragment. These modifications can provide for someadditional property, such as to remove/add amino acids capable ofdisulfide bonding, to increase its bio-longevity, to alter its secretorycharacteristics, etc. In any case, the antibody or antibody fragmentmust possess a bioactive property, such as specific binding to itscognate antigen. Functional or active regions of the antibody orantibody fragment may be identified by mutagenesis of a specific regionof the protein, followed by expression and testing of the expressedpolypeptide. Such methods are readily apparent to a skilled practitionerin the art and can include site-specific mutagenesis of the nucleic acidencoding the antibody or antibody fragment. (Zoller, M. J. Curr. Opin.Biotechnol. 3:348-354, 1992).

As used herein, the term “antibody” or “antibodies” can also refer to ahuman antibody and/or a humanized antibody. Many non-human antibodies(e.g., those derived from mice, rats, or rabbits) are naturallyantigenic in humans, and thus can give rise to undesirable immuneresponses when administered to humans. Therefore, the use of human orhumanized antibodies in the methods of the invention serves to lessenthe chance that an antibody administered to a human will evoke anundesirable immune response.

b) Human Antibodies

The human antibodies of the invention can be prepared using anytechnique. Examples of techniques for human monoclonal antibodyproduction include those described by Cole et al. (Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77, 1985) and by Boemer et al. (J.Immunol., 147(1):86-95, 1991). Human antibodies of the invention (andfragments thereof) can also be produced using phage display libraries(Hoogenboom et al., J. Mol. BioL, 227:381, 1991; Marks et al., J. Mol.Biol., 222:581, 1991).

The human antibodies of the invention can also be obtained fromtransgenic animals. For example, transgenic, mutant mice that arecapable of producing a full repertoire of human antibodies, in responseto immunization, have been described (see, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al.,Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33(1993)). Specifically, the homozygous deletion of the antibody heavychain joining region (J(H)) gene in these chimeric and germ-line mutantmice results in complete inhibition of endogenous antibody production,and the successful transfer of the human germ-line antibody gene arrayinto such germ-line mutant mice results in the production of humanantibodies upon antigen challenge.

c) Humanized Antibodies

Antibody humanization techniques generally involve the use ofrecombinant DNA technology to manipulate the DNA sequence encoding oneor more polypeptide chains of an antibody molecule. Accordingly, ahumanized form of a non-human antibody (or a fragment thereof) is achimeric antibody or antibody chain (or a fragment thereof, such as anFv, Fab, Fab′, or other antigen-binding portion of an antibody) whichcontains a portion of an antigen binding site from a non-human (donor)antibody integrated into the framework of a human (recipient) antibody.

To generate a humanized antibody, residues from one or morecomplementarity determining regions (CDRs) of a recipient (human)antibody molecule are replaced by residues from one or more CDRs of adonor (non-human) antibody molecule that is known to have desiredantigen binding characteristics (e.g., a certain level of specificityand affinity for the target antigen). In some instances, Fv framework(FR) residues of the human antibody are replaced by correspondingnon-human residues. Humanized antibodies may also contain residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.In practice, humanized antibodies are typically human antibodies inwhich some CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies. Humanized antibodiesgenerally contain at least a portion of an antibody constant region(Fc), typically that of a human antibody (Jones et al., Nature,321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), andPresta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.For example, humanized antibodies can be generated according to themethods of Winter and co-workers (Jones et al., Nature, 321:522-525(1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al.,Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Methodsthat can be used to produce humanized antibodies are also described inU.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332(Hoogenboom et al.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No.5,837,243 (Deo et al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.),U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377(Morgan et al.).

d) Administration of Antibodies

Administration of the antibodies can be done as disclosed herein.Nucleic acid approaches for antibody delivery also exist. The broadlyneutralizing anti E7 or Akt antibodies and antibody fragment, or otherantibodies antibodies and antibody fragments disclosed herein, can alsobe administered to patients or subjects as a nucleic acid preparation(e.g., DNA or RNA) that encodes the antibody or antibody fragment, suchthat the patient's or subject's own cells take up the nucleic acid andproduce and secrete the encoded antibody or antibody fragment. Thedelivery of the nucleic acid can be by any means, as disclosed herein,for example.

8. Peptides

a) Protein Variants

As discussed herein there are numerous variants of the E7 protein andAkt protein or the p21^(Cip1) and Raf proteins, for example, that areknown and herein contemplated. In addition, to the known functional E7,Akt, p21^(Cip1), and Raf strain variants, for example, there arederivatives of the E7, Akt, p21^(Cip1), and Raf proteins, for example,which also function in the disclosed methods and compositions. Proteinvariants and derivatives are well understood to those of skill in theart and in can involve amino acid sequence modifications. For example,amino acid sequence modifications typically fall into one or more ofthree classes: substitutional, insertional or deletional variants.Insertions include amino and/or carboxyl terminal fusions as well asintrasequence insertions of single or multiple amino acid residues.Insertions ordinarily will be smaller insertions than those of amino orcarboxyl terminal fusions, for example, on the order of one to fourresidues. Immunogenic fusion protein derivatives, such as thosedescribed in the examples, are made by fusing a polypeptide sufficientlylarge to confer immunogenicity to the target sequence by cross-linkingin vitro or by recombinant cell culture transformed with DNA encodingthe fusion. Deletions are characterized by the removal of one or moreamino acid residues from the protein sequence. Typically, no more thanabout from 2 to 6 residues are deleted at any one site within theprotein molecule. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the protein,thereby producing DNA encoding the variant, and thereafter expressingthe DNA in recombinant cell culture. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, for example M13 primer mutagenesis and PCR mutagenesis. Aminoacid substitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Deletions or insertions preferablyare made in adjacent pairs, i.e. a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof may be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure. Substitutional variants are those in which at least oneresidue has been removed and a different residue inserted in its place.Such substitutions generally are made in accordance with the followingTables 1 and 2 and are referred to as conservative substitutions. TABLE1 Amino Acid Abbreviations Amino Acid Abbreviations Alanine AlaAAllosoleucine AIle Arginine ArgR Asparagines AsnN aspartic acid AspDCysteine CysC glutamic acid GluE Glutamine GlnK Glycine GlyG HistidineHisH Isolelucine IleI Leucine LeuL Lysine LysK Phenylalanine PheFProline ProP pyroglutamic acidp Glu Serine SerS Threonine ThrT TyrosineTyrY Tryptophan TrpW Valine ValV

TABLE 2 Amino Acid Substitutions Exemplary Conservative Substitutions,others are known in the art. Either can be substituted for the other Alaser Ar glys, gln Asn gln; his Asp glu Cys ser Gln asn, lys Glu asp Glyala His asn; gln Ile leu; val Leu ile; val Lys arg; gln; Met Leu; ilePhe met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile; leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table2, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in the proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine, in this case, (e) by increasing the number of sites forsulfation and/or glycosylation.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, lle, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variationsof each explicitly disclosed sequence are included within the mosaicpolypeptides provided herein.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco pp 79-86[1983]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

It is understood that one way to define the variants and derivatives ofthe disclosed proteins herein is through defining the variants andderivatives in terms of homology/identity to specific known sequences.For example, SEQ ID NOs:1-4, 6, 8, and 11 set forth particular sequencesof disclosed proteins. Specifically disclosed are variants of these andother proteins herein disclosed which have at least, 70% or 75% or 80%or 85% or 90% or 95% homology to the stated sequence. Those of skill inthe art readily understand how to determine the homology of twoproteins. For example, the homology can be calculated after aligning thetwo sequences so that the homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment.

It is understood that the description of conservative mutations andhomology can be combined together in any combination, such asembodiments that have at least 70% homology to a particular sequencewherein the variants are conservative mutations.

As this specification discusses various proteins and protein sequencesit is understood that the nucleic acids that can encode those proteinsequences are also disclosed. This would include all degeneratesequences related to a specific protein sequence, i.e. all nucleic acidshaving a sequence that encodes one particular protein sequence as wellas all nucleic acids, including degenerate nucleic acids, encoding thedisclosed variants and derivatives of the protein sequences. Thus, whileeach particular nucleic acid sequence may not be written out herein, itis understood that each and every sequence is in fact disclosed anddescribed herein through the disclosed protein sequence. For example,one of the many nucleic acid sequences that can encode the proteinsequence set foth in SEQ ID NO:8 is set forth in SEQ ID NO:9. Anothernucleic acid sequence that encodes the same protein sequence set forthin SEQ ID NO:8 is set forth in SEQ ID NO:10. In addition, for example, adisclosed conservative derivative of SEQ ID NO:8 is shown in SEQ ID NO:11, where the isoleucine (1) at position 38 is changed to a valine (V).It is understood that for this mutation all of the nucleic acidsequences that encode this particular derivative of the E7 protein arealso disclosed including for example SEQ ID NO:12 and SEQ ID NO:13 whichset forth two of the nucleic acid sequences that encode the particularpolypeptide set forth in SEQ ID NO: I1. It is also understood that whileno amino acid sequence indicates what particular DNA sequence encodesthat protein within an organism, where particular variants of adisclosed protein are disclosed herein, the known nucleic acid sequencethat encodes that protein in the particular strain or species from whichthat protein arises is also known and herein disclosed and described.

It is understood that there are numerous amino acid and peptide analogswhich can be incorporated into the disclosed compositions. For example,there are numerous D amino acids or amino acids which have a differentfunctional substituent then the amino acids shown in Table 1 and Table2. The opposite stereo isomers of naturally occurring peptides aredisclosed, as well as the stereo isomers of peptide analogs. These aminoacids can readily be incorporated into polypeptide chains by chargingtRNA molecules with the amino acid of choice and engineering geneticconstructs that utilize, for example, amber codons, to insert the analogamino acid into a peptide chain in a site specific way (Thorson et al.,Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion inBiotechnology, 3:348-354 (1992); Ibba, Biotechnology & GeneticEnginerring Reviews 13:197-216 (1995), Cahill et al., TIBS,14(10):400403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba andHennecke, Bio/technology, 12:678-682 (1994) all of which are hereinincorporated by reference at least for material related to amino acidanalogs).

Molecules can be produced that resemble peptides, but which are notconnected via a natural peptide linkage. For example, linkages for aminoacids or amino acid analogs can include CH₂NH—, —CH₂S—, —CH₂—CH₂—,—CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CHH₂SO—(These andothers can be found in Spatola, A. F. in Chemistry and Biochemistry ofAmino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker,New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1,Issue 3, Peptide Backbone Modifications (general review); Morley, TrendsPharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res14:177-185 (1979) (—CH₂NR—, CH₂CH₂—); Spatola et al. Life Sci38:1243-1249 (1986) (—CH H₂—S); Hann J. Chem. Soc Perkin Trans. [307-314(1982) (—CH—CH—, cis and trans); Almquist et al. J. Med. Chem.23:1392-1398 (1980) (—COCH₂—); Jennings-White et al. Tetrahedron Lett23:2533 (1982) (—COCH₂—); Szelke et al. European Appln, EP 45665 CA(1982): 97:39405 (1982) (—CH(OH)CH₂—); Holladay et al. Tetrahedron. Lett24:4401-4404 (1983) (—C(OH)CH₂—); and Hruby Life Sci 31:189-199 (1982)(—CH₂—S—); each of which is incorporated herein by reference. Aparticularly preferred non-peptide linkage is —CH₂NH—. It is understoodthat peptide analogs can have more than one atom between the bond atoms,such as b-alanine, g-aminobutyric acid, and the like.

Amino acid analogs and analogs and peptide analogs often have enhancedor desirable properties, such as, more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers.

D-amino acids can be used to generate more stable peptides, because Damino acids are not recognized by peptidases and such. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g., D-lysine in place of L-lysine) canbe used to generate more stable peptides. Cysteine residues can be usedto cyclize or attach two or more peptides together. This can bebeneficial to constrain peptides into particular conformations. (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference).

9. Delivery of the Compositions to Cells

a) Nucleic Acid Delivery

There are a number of compositions and methods which can be used todeliver nucleic acids to cells, either in vitro or in vivo. Thesemethods and compositions can largely be broken down into two classes:viral based delivery systems and non-viral based delivery systems. Forexample, the nucleic acids can be delivered through a number of directdelivery systems such as, electroporation, lipofection, calciumphosphate precipitation, plasmids, viral vectors, viral nucleic acids,phage nucleic acids, phages, cosmids, or via transfer of geneticmaterial in cells or carriers such as cationic liposomes. Appropriatemeans for transfection, including viral vectors, chemical transfectants,or physico-mechanical methods such as electroporation and directdiffusion of DNA, are described by, for example, Wolff, J. A., et al.,Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818,(1991) Such methods are well known in the art and readily adaptable foruse with the compositions and methods described herein. In certaincases, the methods will be modifed to specifically function with largeDNA molecules. Further, these methods can be used to target certaindiseases and cell populations by using the targeting characteristics ofthe carrier.

In the methods described herein, which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), the nucleic acids of the presentinvention can be in the form of naked DNA or RNA, or the nucleic acidscan be in a vector for delivering the nucleic acids to the cells,whereby the encoding DNA or DNA or fragment is under the transcriptionalregulation of a promoter, as would be well understood by one of ordinaryskill in the art as well as enhancers. The vector can be a commerciallyavailable preparation, such as an adenovirus vector (QuantumBiotechnologies, Inc. (Laval, Quebec, Canada).

As one example, vector delivery can be via a viral system, such as aretroviral vector system which can package a recombinant retroviralgenome (see e.g., Pastan et al., Proc. Natl. Acad. Sci. U.S.A. 85:4486,1988; Miller et al., Mol. Cell. Biol. 6:2895, 1986). The recombinantretrovirus can then be used to infect and thereby deliver to theinfected cells nucleic acid encoding a broadly neutralizing antibody (oractive fragment thereof) of the invention. The exact method ofintroducing the altered nucleic acid into mammalian cells is, of course,not limited to the use of retroviral vectors. Other techniques arewidely available for this procedure including the use of adenoviralvectors (Mitani et al., Hum. Gene Ther. 5:941-948, 1994),adeno-associated viral (AAV) vectors (Goodman et al., Blood84:1492-1500, 1994), lentiviral vectors (Naidini et al., Science272:263-267, 1996), pseudotyped retroviral vectors (Agrawal et al.,Exper. Hematol. 24:738-747, 1996). Physical transduction techniques canalso be used, such as liposome delivery and receptor-mediated and otherendocytosis mechanisms (see, for example, Schwartzenberger et al., Blood87:472-478, 1996). This invention can be used in conjunction with any ofthese or other commonly used gene transfer methods.

As one example, if the antibody-encoding nucleic acid or some otherother nucleic acid encoding an inhibitor of the E7 or Akt proteins orencoding a particular variant of the E7 or Akt genes to be used in thedisclosed methods, is delivered to the cells of a subject in anadenovirus vector, the dosage for administration of adenovirus to humanscan range from about 10⁷ to 10⁹ plaque forming units (pfu) per injectionbut can be as high as 10¹² pfu per injection (Crystal, Hum. Gene Ther.8:985-1001, 1997; Alvarez and Curiel, Hum. Gene Ther. 8:597-613, 1997).A subject can receive a single injection, or, if additional injectionsare necessary, they can be repeated at six month intervals (or otherappropriate time intervals, as determined by the skilled practitioner)for an indefinite period and/or until the efficacy of the treatment hasbeen established.

Parenteral administration of the nucleic acid or vector of the presentinvention, if used, is generally characterized by injection. Injectablescan be prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution of suspension in liquidprior to injection, or as emulsions. A more recently revised approachfor parenteral administration involves use of a slow release orsustained release system such that a constant dosage is maintained. See,e.g., U.S. Pat. No. 3,610,795, which is incorporated by referenceherein. For additional discussion of suitable formulations and variousroutes of administration of therapeutic compounds, see, e.g., Remington:The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995.

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome, typically contain integration sequences.These sequences are often viral related sequences, particularly whenviral based systems are used. These viral intergration systems can alsobe incorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of deliver, such as a liposome, so thatthe nucleic acid contained in the delivery system can become integratedinto the host genome.

Other general techniques for integration into the host genome include,for example, systems designed to promote homologous recombination withthe host genome. These systems typically rely on sequence flanking thenucleic acid to be expressed that has enough homology with a targetsequence within the host cell genome that recombination between thevector nucleic acid and the target nucleic acid takes place, causing thedelivered nucleic acid to be integrated into the host genome. Thesesystems and the methods necessary to promote homologous recombinationare known to those of skill in the art.

b) Non-Nucleic Acid Based Systems

The disclosed compositions can be delivered to the target cells in avariety of ways. For example, the compositions can be delivered throughelectroporation, or through lipofection, or through calcium phosphateprecipitation. The delivery mechanism chosen will depend in part on thetype of cell targeted and whether the delivery is occurring for examplein vivo or in vitro.

Thus, the compositions can comprise, in addition to the disclosedcompositions or vectors for example, lipids such as liposomes, such ascationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionicliposomes. Liposomes can further comprise proteins to facilitatetargeting a particular cell, if desired. Administration of a compositioncomprising a compound and a cationic liposome can be administered to theblood afferent to a target organ or inhaled into the respiratory tractto target cells of the respiratory tract. Regarding liposomes, see,e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989);FeIgner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat.No. 4,897,355. Furthermore, the compound can be administered as acomponent of a microcapsule that can be targeted to specific cell types,such as macrophages, or where the diffusion of the compound or deliveryof the compound from the microcapsule is designed for a specific rate ordosage.

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), delivery of the compositions to cells canbe via a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the nucleicacid or vector of this invention can be delivered in vivo byelectroporation, the technology for which is available from Genetronics,Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine(ImaRx Pharmaceutical Corp., Tucson, Ariz.).

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). These techniques can be used for avariety of other specific cell types. Vehicles such as “stealth” andother antibody conjugated liposomes (including lipid mediated drugtargeting to colonic carcinoma), receptor mediated targeting of DNAthrough cell specific ligands, lymphocyte directed tumor targeting, andhighly specific therapeutic retroviral targeting of murine glioma cellsin vivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1 992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

c) In vivo/ex vivo

As described above, the compositions can be administered in apharnmaceutically acceptable carrier and can be delivered to thesubject=s cells in vivo and/or ex vivo by a variety of mechanisms wellknown in the art (e.g., uptake of naked DNA, liposome fusion,intramuscular injection of DNA via a gene gun, endocytosis and thelike).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism, such as, for example, calcium phosphatemediated gene delivery, electroporation, microinjection orproteoliposomes. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

10. Expression Systems

The nucleic acids that are delivered to cells typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain promoters, and/or enhancers tohelp control the expression of the desired gene product. A promoter isgenerally a sequence or sequences of DNA that function when in arelatively fixed location in regard to the transcription start site. Apromoter contains core elements required for basic interaction of RNApolymerase and transcription factors, and may contain upstream elementsand response elements.

a) Viral Promoters and Enhancers

Promoters controlling transcription from vectors in mammalian host cellsmay be obtained from various sources, for example, the genomes ofviruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication (Fiers et al., Nature, 273: 113 (1978)). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HindIII E restriction fragment (Greenway, P. J. et al., Gene 18:355-360 (1982)). Of course, promoters from the host cell or relatedspecies also are useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3′(Lusky, M. L., et al., Mol. Cell Bio. 3: 1108 (1983)) to thetranscription unit. Furthermore, enhancers can be within an intron(Banerdi, J. L. et al., Cell 33: 729 (1983)) as well as within thecoding sequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4: 1293(1984)). They are usually between 10 and 300 bp in length, and theyfunction in cis. Enhancers f unction to increase transcription fromnearby promoters. Enhancers also often contain response elements thatmediate the regulation of transcription. Promoters can also containresponse elements that mediate the regulation of transcription.Enhancers often determine the regulation of expression of a gene. Whilemany enhancer sequences are now known from mammalian genes (globin,elastase, albumin, -fetoprotein and insulin), typically one will use anenhancer from a eukaryotic cell virus for general expression. Preferredexamples are the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

The promotor and/or enhancer may be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone.There are also ways to enhance viral vector gene expression by exposureto irradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

In certain embodiments the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. In certainconstructs the promoter and/or enhancer region be active in alleukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A preferred promoter of this type is theCMV promoter (650 bases). Other preferred promoters are SV40 promoters,cytomegalovirus (full length promoter), and retroviral vector LTF.

It has been shown that all specific regulatory elements can be clonedand used to construct expression vectors that are selectively expressedin specific cell types such as melanoma cells. The glial fibrillaryacetic protein (GFAP) promoter has been used to selectively expressgenes in cells of glial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contain a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

b) Markers

The viral vectors can include nucleic acid sequence encoding a markerproduct. This marker product is used to determine if the gene has beendelivered to the cell and once delivered is being expressed. Preferredmarker genes are the E. Coli lacZ gene, which encodes β-galactosidase,and green fluorescent protein.

In some embodiments the marker may be a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hydromycin, and puromycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Twoexamples are: CHO DHFR- cells and mouse LTK-cells. These cells lack theability to grow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern P. and Berg,P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan,R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B.et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employbacterial genes under eukaryotic control to convey resistance to theappropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid)or hygromycin, respectively. Others include the neomycin analog G418 andpuramycin.

11. Pharmaceutical Carriers/Delivery of Pharmaceutical Products

As described above, the compositions can also be administered in vivo ina pharmaceutically acceptable carrier. By “pharmaceutically acceptable”is meant a material that is not biologically or otherwise undesirable,i.e., the material may be administered to a subject, along with thenucleic acid or vector, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

The compositions may be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, topically or the like,although topical intranasal administration or administration by inhalantis typically preferred. As used herein, “topical intranasaladministration” means delivery of the compositions into the nose andnasal passages through one or both of the nares and can comprisedelivery by a spraying mechanism or droplet mechanism, or throughaerosolization of the nucleic acid or vector. The latter may beeffective when a large number of animals is to be treatedsimultaneously. Administration of the compositions by inhalant can bethrough the nose or mouth via delivery by a spraying or dropletmechanism. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of thecompositions required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the allergic disorder being treated, the particular nucleicacid or vector used, its mode of administration and the like. Thus, itis not possible to specify an exact amount for every composition.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and otherantibody conjugated liposomes (including lipid mediated drug targetingto colonic carcinoma), receptor mediated targeting of DNA through cellspecific ligands, lymphocyte directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells invivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research,49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

a) Pharmaceutically Acceptable Carriers

The compositions, including antibodies, can be used therapeutically incombination with a pharmaceutically acceptable carrier.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceu Formulations for topical administration may include ointments,lotions, creams, gels, drops, suppositories, sprays, liquids andpowders. Conventional pharmaceutical carriers, aqueous, powder or oilybases, thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

b) Therapeutic Uses

The dosage ranges for the administration of the compositions are thoselarge enough to produce the desired effect in which the symptomsdisorder are effected. The dosage should not be so large as to causeadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage will vary with the age,condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The dosage can be adjusted by theindividual physician in the event of any counterindications. Dosage canvary, and can be administered in one or more dose administrations daily,for one or several days.

12. Chips and Micro Arrays

Disclosed are chips where at least one address is the sequences or partof the sequences set forth in any of the nucleic acid sequencesdisclosed herein. Also disclosed are chips where at least one address isthe sequences or portion of sequences set forth in any of the peptidesequences disclosed herein.

Also disclosed are chips where at least one address is a variant of thesequences or part of the sequences set forth in any of the nucleic acidsequences disclosed herein. Also disclosed are chips where at least oneaddress is a variant of the sequences or portion of sequences set forthin any of the peptide sequences disclosed herein.

Disclosed are chips comprising E7 protein and Akt protein or thep21^(Cip1) and Raf protein (or nucleic acid related to the same) whichare designed such that the chips can be used to identify moleculeshaving the properties disclosed herein. Also disclosed are chips wherethe chips comprise molecules that interact with either E7 protein andAkt protein or the p21^(Cip1) and Raf protein.

13. Computer Readable Mediums

It is understood that the disclosed nucleic acids and proteins can berepresented as a sequence consisting of the nucleotides of amino acids,and that the relationships between protein molecules can be stored in acomputer readable medium. There are a variety of ways to display thesesequences or molecule relationships, such as the relationship betweenE7, Akt, Raf, and p21^(Cip1), for example the nucleotide guanosine canbe represented by G or g and the amino acid valine can be represented byVal or V, and the proteins themselves could be stored as, for example,their sequence or by representations of the protein, such as a definingword or symbol. Those of skill in the art understand how to display andexpress any nucleic acid or protein sequence or protein relationship inany of the variety of ways that exist, each of which is consideredherein disclosed. Specifically contemplated herein is the display orstorage of these sequences or molecule relationships on computerreadable mediums, such as, commercially available floppy disks, tapes,chips, hard drives, compact disks, and video disks, or other computerreadable mediums. Also disclosed are the binary code representations ofthe disclosed sequences and the disclosed molecule relationships. Thoseof skill in the art understand what computer readable mediums. Thus,computer readable mediums on which the nucleic acids or proteinsequences are recorded, stored, or saved.

Disclosed are computer readable mediums comprising the sequences and/ormolecule relationships and information regarding the sequences and/ormolecule relationships set forth herein, as well as the relationshipsbetween the proteins disclosed in the mechanisms disclosed herein.

14. Kits

Disclosed herein are kits that are drawn to reagents that can be used inpracticing the methods disclosed herein. The kits can include anyreagent or combination of reagent discussed herein or that would beunderstood to be required or beneficial in the practice of the disclosedmethods. For example, the kits could include primers to perform theamplification reactions discussed in certain embodiments of the methods,as well as the buffers and enzymes required to use the primers asintended. The kits could also include, for example, cells that aredeisigned as disclosed herein, for use in screening or testing theactivity of compounds that modulate the effect of E7 on a cell.

C. METHODS OF MAKING THE COMPOSITIONS

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted.

1. Nucleic Acid Synthesis

For example, the nucleic acids, such as, the oligonucleotides to be usedas primers can be made using standard chemical synthesis methods or canbe produced using enzymatic methods or any other known method. Suchmethods can range from standard enzymatic digestion followed bynucleotide fragment isolation (see for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) topurely synthetic methods, for example, by the cyanoethyl phosphoramiditemethod using a Milligen or Beckman System 1Plus DNA synthesizer (forexample, Model 8700 automated synthesizer of Milligen-Biosearch,Burlington, Mass. or ABI Model 380B). Synthetic methods useful formaking oligonucleotides are also described by Ikuta et al., Ann. Rev.Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triestermethods), and Narang et al., Methods Enzymol., 65:610-620 (1980),(phosphotriester method). Protein nucleic acid molecules can be madeusing known methods such as those described by Nielsen et al.,Bioconjug. Chem. 5:3-7 (1994).

2. Peptide Synthesis

One method of producing the disclosed proteins, such as SEQ, is to linktwo or more peptides or polypeptides together by protein chemistrytechniques. For example, peptides or polypeptides can be chemicallysynthesized using currently available laboratory equipment using eitherFmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl)chemistry. (Applied Biosystems, Inc., Foster City, Calif.). One skilledin the art can readily appreciate that a peptide or polypeptidecorresponding to the disclosed proteins, for example, can be synthesizedby standard chemical reactions. For example, a peptide or polypeptidecan be synthesized and not cleaved from its synthesis resin whereas theother fragment of a peptide or protein can be synthesized andsubsequently cleaved from the resin, thereby exposing a terminal groupwhich is functionally blocked on the other fragment. By peptidecondensation reactions, these two fragments can be covalently joined viaa peptide bond at their carboxyl and amino termini, respectively, toform an antibody, or fragment thereof. (Grant G A (1992) SyntheticPeptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky Mand Trost B., Ed. (1 993) Principles of Peptide Synthesis.Springer-Verlag Inc., NY (which is herein incorporated by reference atleast for material related to peptide synthesis). Alternatively, thepeptide or polypeptide is independently synthesized in vivo as describedherein. Once isolated, these independent peptides or polypeptides may belinked to form a peptide or fragment thereof via similar peptidecondensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains (Abrahmsen L etal., Biochemistry, 30:4151 (1991)). Alternatively, native chemicalligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson et al.Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779(1994)). The first step is the chemoselective reaction of an unprotectedsynthetic peptide—thioester with another unprotected peptide segmentcontaining an amino-terminal Cys residue to give a thioester-linkedintermediate as the initial covalent product. Without a change in thereaction conditions, this intermediate undergoes spontaneous, rapidintramolecular reaction to form a native peptide bond at the ligationsite (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I etal., J.Biol.Chem., 269:16075 (1994); Clark-Lewis I et al., Biochemistry,30:3128 (1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).

Alternatively, unprotected peptide segments are chemically linked wherethe bond formed between the peptide segments as a result of the chemicalligation is an unnatural (non-peptide) bond (Schnolzer, M et al.Science, 256:221 (1992)). This technique has been used to synthesizeanalogs of protein domains as well as large amounts of relatively pureproteins with full biological activity (deLisle Milton R C et al.,Techniques in Protein Chemistry IV. Academic Press, New York, pp.257-267 (1992)).

3. Process for Making the Compositions

Disclosed are processes for making the compositions as well as makingthe intermediates leading to the compositions. There are a variety ofmethods that can be used for making these compositions, such assynthetic chemical methods and standard molecular biology methods. It isunderstood that the methods of making these and the other disclosedcompositions are specifically disclosed.

Disclosed are cells produced by the process of transforming the cellwith any of the disclosed nucleic acids. Disclosed are cells produced bythe process of transforming the cell with any of the non-naturallyoccurring disclosed nucleic acids.

Disclosed are any of the disclosed peptides produced by the process ofexpressing any of the disclosed nucleic acids. Disclosed are any of thenon-naturally occurring disclosed peptides produced by the process ofexpressing any of the disclosed nucleic acids. Disclosed are any of thedisclosed peptides produced by the process of expressing any of thenon-naturally disclosed nucleic acids.

Disclosed are animals produced by the process of transfecting a cellwithin the animal with any of the nucleic acid molecules disclosedherein. Disclosed are animals produced by the process of transfecting acell within the animal any of the nucleic acid molecules disclosedherein, wherein the animal is a mammal. Also disclosed are animalsproduced by the process of transfecting a cell within the animal any ofthe nucleic acid molecules disclosed herein, wherein the mammal ismouse, rat, rabbit, cow, sheep, pig, or primate.

Also disclosed are animals produced by the process of adding to theanimal any of the cells disclosed herein.

Disclosed are methods of making a composition capable of inhibiting E7cellular proliferation activity comprising mixing an E7 inhibitingcompound with a pharmaceutically acceptable carrier, wherein thecompound is identified, or can be identified, by administering thecompound to a system, wherein the system causes maintenance of Akt,assaying the effect of the compound on the amount of Akt in the system,and selecting a compound which causes a decrease in the amount of Aktpresent in the system.

Disclosed are methods of making a compound that inhibits E7 cellularproliferation activity comprising, a) administering a compound to asystem, wherein the system causes maintenance of Akt, b) assaying theeffect of the compound on the amount of Akt in the system, c) selectinga compound which causes a decrease in the amount of Akt present in thesystem, and d) synthesizing the compound.

Disclosed are methods of making a composition capable of inhibiting E7Akt maintenance activity comprising mixing the compound with apharmaceutical carrier and wherein the compound is identified, or can beidentified, by administering the compound to a system, wherein thesystem comprises E7 Akt maintenance activity, assaying the effect of thecompound on E7 Akt maintenance activity, and selecting a compound whichinhibits E7 Akt maintenance activity.

Disclosed are methods of making a compound capable of reversing theeffect E7 has on Akt comprising, a) administering a compound to asystem, wherein the system comprises E7 Akt maintenance activity, b)assaying the effect of the compound on E7 Akt maintenance activity, c)selecting a compound which inhibits E7 Akt maintenance activity, and d)synthesizing the compound.

It is understood that the disclosed methods can also be performed wherethe amount of active, i.e. phosphorylated Akt, is present are assayed.It is also understood that the disclosed methods can be performed bylooking at the activity of Akt, for example, the phosphorylation ofp21^(Cip).

D. METHODS OF USING THE COMPOSITIONS

The disclosed compositions can be used in a variety of ways as researchtools. For example, the disclosed compositions can be used to identifymolecule that modulate the effect E7 has on the cell through E7'smodulation of Akt activity.

1. Methods of Treatment

The disclosed compositions can be used to treat any disease whereuncontrolled cellular proliferation occurs such as cancers, typicallycancers where uncontrolled proliferation involves E7 or E7 variants orAKT or AKT variants. Cancers that are associated with HPV can be targetcancers. For example, Penile, vaginal, and vulvar cancers are associatedwith HPVs. A non-limiting list of different types of cancers is asfollows: lymphomas (Hodgkins and non-Hodgkins), leukemias, carcinomas,carcinomas of solid tissues, squamous cell carcinomas, adenocarcinomas,sarcomas, gliomas, high grade gliomas, blastomas, neuroblastomas,plasmacytomas, histiocytomas, melanomas, adenomas, hypoxic tumours,myelomas, AIDS-related lymphomas or sarcomas, metastatic cancers, orcancers in general.

A representative but non-limiting list of cancers that the disclosedcompositions can be used to treat is the following: lymphoma, B celllymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloidleukemia, bladder cancer, brain cancer, nervous system cancer, head andneck cancer, squamous cell carcinoma of head and neck, kidney cancer,lung cancers such as small cell lung cancer and non-small cell lungcancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer,prostate cancer, skin cancer, liver cancer, melanoma, squamous cellcarcinomas of the mouth, throat, larynx, and lung, colon cancer,cervical cancer, cervical carcinoma, breast cancer, and epithelialcancer, renal cancer, genitourinary cancer, pulmonary cancer, esophagealcarcinoma, head and neck carcinoma, large bowel cancer, hematopoieticcancers; testicular cancer; colon and rectal cancers, prostatic cancer,or pancreatic cancer.

Compounds disclosed herein may also be used for the treatment ofprecancer conditions such as cervical and anal dysplasias, otherdysplasias, severe dysplasias, hyperplasias, atypical hyperplasias, andneoplasias.

Disclosed are methods of inhibiting aberrant cellular proliferationcomprising, administering a compound which inhibits E7 Akt maintenanceactivity.

Also disclosed are methods of inhibiting abberant cellularproliferation, wherein administering the compound occurs in a subject orwherein the subject is a subject who has cancer.

Also disclosed are methods of inhibiting E7 cellular proliferationactivity comprising administering a compound, wherein the compoundcauses degradation of active Akt, wherein the compound is defined as acompound capable of being identified by administering the compound to asystem, wherein the system causes maintenance of active Akt, assayingthe effect of the compound on the amount of active Akt in the system,and selecting a compound which causes a decrease in the amount of activeAkt present in the system.

Disclosed are methods of inhibiting E7 cellular proliferation activitycomprising administering a compound that causes the degradation ofactive Akt.

Disclosed are methods of inhibiting E7 cellular proliferation activitycomprising administering a compound, wherein the compound is identified,or canbe identified, as maintaining Akt activity.

Disclosed are methods of inhibiting E7 cellular proliferation activitycomprising administering an inhibitor of E7 Akt maintenance activity,wherein the inhibitor is a compound capable of being identified byadministering the compound to a system, wherein the system comprises E7Akt maintenance activity, assaying the effect of the compound on E7 Aktmaintenance activity, and selecting a compound which inhibits E7 Aktmaintenance activity.

Disclosed are methods of inhibiting E7 cellular proliferation activitycomprising administering an inhibitor of E7 Akt maintenance activity.

Disclosed are methods of inhibiting E7 cellular proliferation activitycomprising administering a compound, wherein the compound is identified,or can be identified, as inhibiting E7 Akt maintenance activity.

It is understood that all of the disclosed methods for identifying thevarious compounds and compositions discussed herein can also representmethods of inhibiting the vartious E7 effects as well as methods ofmaking the various compounds and compositions identified.

It is understood that the disclosed methods can also be performed wherethe amount of active, i.e. phosphorylated Akt, is present are assayed.It is also understood that the disclosed methods can be performed bylooking at the activity of Akt, for example, the phosphorylation ofp21^(Cip).

2. Methods of Identifying Modulators of the E7 Affect on Akt and onp21^(Cip)

The disclosed systems can be used to identify compositions that modulatethe effect of E7 on Akt, for example.

Disclosed are methods of identifying a compound that inhibits E7cellular proliferation activity comprising, a) administering a compoundto a system, wherein the system causes maintenance of Akt activity; b)assaying the effect of the compound on the amount of Akt activity in thesystem; and c) selecting a compound which causes a decrease in theamount of Akt activity present in the system.

Disclosed are methods of identifying a compound capable of reversing theeffect E7 has on Akt comprising, a) administering a compound to asystem, wherein the system comprises E7 active Akt maintenance activity,b) assaying the effect of the compound on E7 active Akt maintenanceactivity, and c) selecting a compound which inhibits E7 active Aktmaintenance activity.

Disclosed are methods of identifying a compound which promotes thenuclear localization of p21^(Cip1) comprising, a) administering acompound to a system, wherein the system comprises E7 p21^(Cip1)cytoplasmic localization activity, b) assaying the effect of thecompound on E7 p21^(Cip1) cytoplasmic localization activity, and c)selecting a compound which promotes p21^(Cip1) nuclear localizationactivity.

Disclosed are methods of promoting p21^(Cip1) nuclear localization,comprising a) administering a compound to a system, wherein the systemcomprises E7 p21^(Cip1) cytoplasmic localization activity, b) assayingthe effect of the compound on E7 p21^(Cip1) cytoplasmic localizationactivity, and c) selecting a compound which promotes p21^(Cip1) nuclearlocalization activity.

Disclosed are methods of identifying an inhibitor of an interactionbetween Akt and E7 comprising a) administering a compound to a system,wherein the system comprises E7, b) assaying the effect of the compoundon an E7-Akt interaction, and c) selecting a compound which inhibits E7Akt interaction.

It is understood that the disclosed methods and systems for identifyingmolecules having the E7 effect inhibiting properties disclosed hereincan be coupled with combinatorial chemistry protocols and concepts. Adiscussion of combinatorial chemistry and general methods and conceptsis discussed herein as well as computer assisted composition design. Forexample, molecules that are identified using in vitro combinatorialchemistry methods can then be assayed for appropriate activity in thedisclosed system. The systems could also for example, be used in aninterative screening process using many potential inhibitors asdiscussed herein.

It is understood that the disclosed methods can also be performed wherethe amount of active, i.e. phosphorylated Akt, is present are assayed.It is also understood that the disclosed methods can be performed bylooking at the activity of Akt, for example, the phosphorylation ofp21^(Cip).

a) Compositions Identified by Screening with DisclosedCompositions/Combinatorial Chemistry

(1) Combinatorial Chemistry

The disclosed compositions and relationships can be used as targets forany combinatorial technique to identify molecules or macromolecularmolecules that interact with the disclosed compositions and effect therelationships in a desired way. Also disclosed are the compositions thatare identified through combinatorial techniques or screening techniquesin which E7 or Akt or portions thereof, for example, are used as thetarget in a combinatorial or screening protocol.

It is understood that when using the disclosed compositions incombinatorial techniques or screening methods, molecules, such asmacromolecular molecules, will be identified that have particulardesired properties such as inhibition or stimulation or the targetmolecule's function. The molecules identified and isolated when usingthe disclosed compositions, such as, E7 or Akt, for example, or thecells disclosed herein, are also disclosed. Thus, the products producedusing the combinatorial or screening approaches that involve thedisclosed compositions, such as, E7, Akt, or the cells disclosed arealso considered herein disclosed.

Combinatorial chemistry includes but is not limited to all methods forisolating small molecules or macromolecules that are capable of bindingeither a small molecule or another macromolecule, typically in aniterative process. Proteins, oligonucleotides, and sugars are examplesof macromolecules. For example, oligonucleotide molecules with a givenfunction, catalytic or ligand-binding, can be isolated from a complexmixture of random oligonucleotides in what has been referred to as “invitro genetics” (Szostak, TIBS 19:89, 1992). One synthesizes a largepool of molecules bearing random and defined sequences and subjects thatcomplex mixture, for example, approximately 10¹⁵ individual sequences in100 μg of a 100 nucleotide RNA, to some selection and enrichmentprocess. Through repeated cycles of affinity chromatography and PCRamplification of the molecules bound to the ligand on the column,Ellington and Szostak (1990) estimated that 1 in 10¹⁰ RNA moleculesfolded in such a way as to bind a small molecule dyes. DNA moleculeswith such ligand-binding behavior have been isolated as well (Ellingtonand Szostak, 1992; Bock et al, 1992). Techniques aimed at similar goalsexist for small organic molecules, proteins, antibodies and othermacromolecules known to those of skill in the art. Screening sets ofmolecules for a desired activity whether based on small organiclibraries, oligonucleotides, or antibodies is broadly referred to ascombinatorial chemistry. Combinatorial techniques are particularlysuited for defining binding interactions between molecules and forisolating molecules that have a specific binding activity, often calledaptamers when the macromolecules are nucleic acids.

There are a number of methods for isolating proteins which either havede novo activity or a modified activity. For example, phage displaylibraries have been used to isolate numerous peptides that interact witha specific target. (See for example, U.S. Pat. No. 6,031,071; 5,824,520;5,596,079; and 5,565,332 which are herein incorporated by reference atleast for their material related to phage display and methods relate tocombinatorial chemistry)

A preferred method for isolating proteins that have a given function isdescribed by Roberts and Szostak (Roberts R. W. and Szostak J. W. Proc.Natl. Acad. Sci. USA, 94(23)12997-302 (1997). This combinatorialchemistry method couples the functional power of proteins and thegenetic power of nucleic acids. An RNA molecule is generated in which apuromycin molecule is covalently attached to the 3′-end of the RNAmolecule. An in vitro translation of this modified RNA molecule causesthe correct protein, encoded by the RNA to be translated. In addition,because of the attachment of the puromycin, a peptdyl acceptor whichcannot be extended, the growing peptide chain is attached to thepuromycin which is attached to the RNA. Thus, the protein molecule isattached to the genetic material that encodes it. Normal in vitroselection procedures can now be done to isolate functional peptides.Once the selection procedure for peptide function is completetraditional nucleic acid manipulation procedures are performed toamplify the nucleic acid that codes for the selected functionalpeptides. After amplification of the genetic material, new RNA istranscribed with puromycin at the 3′-end, new peptide is translated andanother functional round of selection is performed. Thus, proteinselection can be performed in an iterative manner just like nucleic acidselection techniques. The peptide which is translated is controlled bythe sequence of the RNA attached to the puromycin. This sequence can beanything from a random sequence engineered for optimum translation (i.e.no stop codons etc.) or it can be a degenerate sequence of a known RNAmolecule to look for improved or altered function of a known peptide.The conditions for nucleic acid amplification and in vitro translationare well known to those of ordinary skill in the art and are preferablyperformed as in Roberts and Szostak (Roberts R. W. and Szostak J. W.Proc. Natl. Acad. Sci. USA, 94(23)12997-302 (1997)).

Another preferred method for combinatorial methods designed to isolatepeptides is described in Cohen et al. (Cohen B.A.,et al., Proc. NatI.Acad. Sci. USA 95(24): 14272-7 (1998)). This method utilizes andmodifies two-hybrid technology. Yeast two-hybrid systems are useful forthe detection and analysis of protein:protein interactions. Thetwo-hybrid system, initially described in the yeast Saccharomycescerevisiae, is a powerful molecular genetic technique for identifyingnew regulatory molecules, specific to the protein of interest (Fieldsand Song, Nature 340:245-6 (1989)). Cohen et al., modified thistechnology so that novel interactions between synthetic or engineeredpeptide sequences could be identified which bind a molecule of choice.The benefit of this type of technology is that the selection is done inan intracellular environment. The method utilizes a library of peptidemolecules that attached to an acidic activation domain. A peptide ofchoice, for example a portion of AKT OR E7 is attached to a DNA bindingdomain of a transcriptional activation protein, such as Gal 4. Byperforming the Two-hybrid technique on this type of system, moleculesthat bind the portion of Akt or E7 can be identified.

Using methodology well known to those of skill in the art, incombination with various combinatorial libraries, one can isolate andcharacterize those small molecules or macromolecules, which bind to orinteract with the desired target. The relative binding affinity of thesecompounds can be compared and optimum compounds identified usingcompetitive binding studies, which are well known to those of skill inthe art.

Techniques for making combinatorial libraries and screeningcombinatorial libraries to isolate molecules which bind a desired targetare well known to those of skill in the art. Representative techniquesand methods can be found in but are not limited to U.S. Pat. Nos.5,084,824, 5,288,514, 5,449,754, 5,506,337, 5,539,083, 5,545,568,5,556,762, 5,565,324, 5,565,332, 5,573,905, 5,618,825, 5,619,680,5,627,210, 5,646,285, 5,663,046, 5,670,326, 5,677,195, 5,683,899,5,688,696, 5,688,997, 5,698,685, 5,712,146, 5,721,099,5,723,598,5,741,713, 5,792,431, 5,807,683, 5,807,754, 5,821,130,5,831,014, 5,834,195, 5,834,318,5,834,588, 5,840,500, 5,847,150,5,856,107, 5,856,496, 5,859,190, 5,864,010, 5,874,443, 5,877,214,5,880,972, 5,886,126, 5,886,127, 5,891,737, 5,916,899, 5,919,955,5,925,527, 5,939,268, 5,942,387, 5,945,070, 5,948,696, 5,958,702,5,958,792, 5,962,337, 5,965,719,5,972,719, 5,976,894, 5,980,704,5,985,356, 5,999,086, 6,001,579, 6,004,617, 6,008,321, 6,017,768,6,025,371, 6,030,917, 6,040,193, 6,045,671, 6,045,755, 6,060,596, and6,061,636.

Combinatorial libraries can be made from a wide array of molecules usinga number of different synthetic techniques. For example, librariescontaining fused 2,4-pyrimidinediones (U.S. Pat. No. 6,025,371)dihydrobenzopyrans (U.S. Pat. Nos. 6,017,768and 5,821,130), amidealcohols (U.S. Pat. No. 5,976,894), hydroxy-amino acid amides (U.S. Pat.No. 5,972,719) carbohydrates (U.S. Pat. No. 5,965,719),1,4-benzodiazepin-2,5-diones (U.S. Pat. No. 5,962,337), cyclics (U.S.Pat. No. 5,958,792), biaryl amino acid amides (U.S. Pat. No. 5,948,696),thiophenes (U.S. Pat. No. 5,942,387), tricyclic Tetrahydroquinolines(U.S. Pat. No. 5,925,527), benzofurans (U.S. Pat. No. 5,919,955),isoquinolines (U.S. Pat. No. 5,916,899), hydantoin and thiohydantoin(U.S. Pat. No. 5,859,190), indoles (U.S. Pat. No. 5,856,496),imidazol-pyrido-indole and imidazol-pyrido-benzothiophenes (U.S. Pat.No. 5,856,107) substituted 2-methylene-2,3-dihydrothiazoles (U.S. Pat.No. 5,847,150), quinolines (U.S. Pat. No. 5,840,500), PNA (U.S. Pat. No.5,831,014), containing tags (U.S. Pat. No. 5,721,099), polyketides (U.S.Pat. No. 5,712,146), morpholino-subunits (U.S. Pat. Nos. 5,698,685 and5,506,337), sulfamides (U.S. Pat. No. 5,618,825), and benzodiazepines(U.S. Pat. No. 5,288,514).

As used herein combinatorial methods and libraries included traditionalscreening methods and libraries as well as methods and libraries used ininterative processes.

(2) Computer Assisted Drug Design

The disclosed compositions can be used as targets for any molecularmodeling technique to identify either the structure of the disclosedcompositions or to identify potential or actual molecules, such as smallmolecules, which interact in a desired way with the disclosedcompositions.

It is understood that when using the disclosed compositions in modelingtechniques, molecules, such as macromolecular molecules, will beidentified that have particular desired properties such as inhibition orstimulation or the target molecule's function. The molecules identifiedand isolated when using the disclosed compositions, such as, E7 or Aktor the cells disclosed herein, are also disclosed. Thus, the productsproduced using the combinatorial or screening approaches that involvethe disclosed compositions, such as, E7, Akt, or the cells disclosed arealso considered herein disclosed.

Thus, one way to isolate molecules that bind a molecule of choice isthrough rational design. This is achieved through structural informationand computer modeling. Computer modeling technology allows visualizationof the three-dimensional atomic structure of a selected molecule and therational design of new compounds that will interact with the molecule.The three-dimensional construct typically depends on data from x-raycrystallographic analyses or NMR imaging of the selected molecule. Themolecular dynamics require force field data. The computer graphicssystems enable prediction of how a new compound will link to the targetmolecule and allow experimental manipulation of the structures of thecompound and target molecule to perfect binding specificity. Predictionof what the molecule-compound interaction will be when small changes aremade in one or both requires molecular mechanics software andcomputationally intensive computers, usually coupled with user-friendly,menu-driven interfaces between the molecular design program and theuser.

Examples of molecular modeling systems are the CHARMm and QUANTAprograms, Polygen Corporation, Waltham, Mass. CHARMm performs the energyminimization and molecular dynamics functions. QUANTA performs theconstruction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive withspecific proteins, such as Rotivinen, et al., 1988 Acda PharmaceuticaFennica 97, 159-166; Ripka, New Scientist 54-57 (Jun. 16, 1988);McKinaly and Rossmann, 1989 Annu. Rev. Pharinacol._Toxiciol. 29,111-122; Perry and Davies, QSAR: Ouantitative Structure-ActivityRelationships in Drug Design pp. 189-193 (Alan R. Liss, Inc. 1989);Lewis and Dean, 1989 Proc. R. Soc. Lond. 236, 125-140 and 141-1 62; and,with respect to a model enzyme for nucleic acid components, Askew, etal., 1989 J. Am. Chem. Soc. 111, 1082-1090. Other computer programs thatscreen and graphically depict chemicals are available from companiessuch as BioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga,Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario. Although theseare primarily designed for application to drugs specific to particularproteins, they can be adapted to design of molecules specificallyinteracting with specific regions of DNA or RNA, once that region isidentified.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichalter substrate binding or enzymatic activity.

E. EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in IC or is atambient temperature, and pressure is at or near atmospheric.

1. Example 1

a) Results

(1) E7 Overcomes Raf-Induced Growth Arrest.

The ability of HPV16 E7 to abrogate p21^(Cip1)-dependent growth arresthas been observed, but the targets of E7 to propogate this effect aredisclosed herein. Activation of the Ras/Raf/MAPK pathway induces cellcycle arrest in various primary and immortal cell types (Hirakawa andRuley, 1988; Lloyd et al., 1997; Ridley et al., 1988; Serrano et al.,1997; Sewing et al., 1997; Woods et al., 1997). Importantly, biochemicaland genetic approaches indicate that p21^(Cip1) is an essential mediatorof this arrest phenotype. To explore the molecular basis for E7-mediatedbypass of Ras-induced arrest, a conditional RafAR molecule in which anactivated Raf kinase has been fused to the androgen receptorhormone-binding domain was used (Sewing, A., B. Wiseman, A. C. Lloyd,and H. Land 1997. High-intensity Raf signal causes cell cycle arrestmediated by p21Cip1 Molecular & Cellular Biology. 17:5588-97).Importantly, Raf acts directly downstream of Ras ( Moodie, S. A., B. M.Willumsen, M. J. Weber, and A. Wolfman 1993. Complexes of Ras.GTP withRaf-1 and mitogen-activated protein kinase kinase Science. 260:1658-61,Vojtek, A. B., S. M. Hollenberg, and J. A. Cooper 1993. Mammalian Rasinteracts directly with the serine/threonine kinase Raf Cell.74:205-14.), and Ras effector loop mutants that preferentially activateRaf (but not PI(3)K or Ral.GDS) confer growth arrest similar tooncogenic Ras (Lin, A. W., M. Barradas, J. C. Stone, L. van Aelst, M.Serrano, and S. W. Lowe 1998. Premature senescence involving p53 and p16is activated in response to constitutive MEK/MAPK mitogenic signalingGenes & Development. 12:3008-19.). Additionally, Raf renders similargrowth and morphological phenotypes as Ras in primary and immortalfibroblasts (Lin, A. W., M. Barradas, J. C. Stone, L. van Aelst, M.Serrano, and S. W. Lowe 1998. Premature senescence involving p53 and p16is activated in response to constitutive MEK/MAPK mitogenic signalingGenes & Development. 12:3008-19., Sewing, A., B. Wiseman, A. C. Lloyd,and H. Land 1997. High-intensity Raf signal causes cell cycle arrestmediated by p21Cip1 Molecular & Cellular Biology. 17:5588-97). In NIH3T3fibroblasts, activation of a steroid hormone regulatable RafAR kinaseleads to inhibition of DNA synthesis preceded by increased expression ofp21^(Cip1) and loss of cyclin E-CDK2 kinase activity (Sewing et al.,1997). As such, this cell-based system represents a relevant contextwithin which to examine the interplay between E7 and p21^(Cip1). Inorder to assess the effects of E7 in this system, RafAR-expressingNIH3T3 were infected with the retroviral vector pBabe (Morgenstem andLand, 1990) or its derivative encoding HPV16 E7. Infected cells werepooled, examined for E7 expression (FIG. 1A), and used for subsequentexperiments. Activation of RafAR with 1.0 μM R1881 led to morphologicalchanges including elongation and development of extended processes (datanot shown). These RafAR-induced alterations in cell morphology, whichare consistent with previous descriptions in NIH3T3 and other cell types(Lloyd et al., 1997; Sewing et al., 1997), were not affected inE7-expressing cells, indicating that at least some components of Rafsignaling are not disrupted by the presence of E7. Upon examination ofcell proliferation, RafAR activation led to G1 arrest in control cells(Babe), with >85% of cells accumulating in G1 (FIG. 1B). This wasaccompanied by inhibition of DNA synthesis as shown in FIG. 1C. However,cells expressing E7 continued cell cycle progression (FIG. 1B) and DNAsynthesis (FIG. 1C) in the presence of activated RafAR. Similarobservations have been made in separately generated clonal and pooledE7-expressing cell lines. These results suggest that E7 perturbsRaf-induced negative regulation at the G1-S transition and are inagreement with previous observations that E7 transforms cells incooperation with an activated Ras/Raf pathway (Halpem, 1997).

(2) E7 Prevents Inactivation of Cyclin E-CDK2 by p21^(Cip1)

In NIH3T3 fibroblasts, Raf activation leads to inhibition of DNAsynthesis preceded by increased expression of p21^(Cip1) and loss ofcyclin E-CDK2 kinase activity (Sewing, A., B. Wiseman, A. C. Lloyd, andH. Land 1997. High-intensity Raf signal causes cell cycle arrestmediated by p21Cip1 Molecular & Cellular Biology. 17:5588-97, Woods, D.,D. Parry, H. Cherwinski, E. Bosch, E. Lees, and M. McMahon 1997.Raf-induced proliferation or cell cycle arrest is determined by thelevel of Raf activity with arrest mediated by p21Cip1 Molecular &Cellular Biology. 17:5598-611.). To detennine the nature of resistanceto RafAR-induced arrest in E7-expressing cells, the expression levelsand activities of G 1-specific cyclins, CDKs, and CKIs were examined.Activation of RafAR led to induction of cyclin D1 and cyclin E incontrol and E7-expressing cells (FIG. 2A). Importantly, p21^(Cip1) wasalso elevated in both cell lines upon RafAR stimulation (FIG. 2A).Consistent with observations in other p21^(Cip1)-dependent arrestsystems (Funk et al., 1997; Jones et al., 1997; Ruesch and Laimins,1997), this implies that E7 does not overcome p21^(Cip1)-mediated arrestby preventing p21^(Cip1) expression. In RafAR-arrested control cells,stimulation of RafAR resulted in the loss of steady state cyclin A andaccumulation of hypophosphorylated RB (FIG. 2A). Cyclin E-CDK2 activityis required for hyperphosphorylation of RB and cyclin A expression(Rudolph et al., 1996; Weinberg, 1995; Zerfass-Thome et al., 1997). Inaccordance, control cells exhibited significant loss of cyclin E-CDK2activity and a less dramatic decrease in CDK4-associated kinase activity(FIG. 2B and FIG. 2C), likely due to a greater sensitivity of CDK2 toinhibition by Kip/Cip CKIs (Cheng et al., 1999; LaBaer et al., 1997;Polyak et al., 1994b). In contrast, cyclin E-CDK2 and CDK4 kinaseactivities were maintained upon RafAR stimulation of E7-expressing cells(FIG. 2B and FIG. 2C). In addition, cyclin A expression and RBhyperphosphorylation were similar in asynchronous and RafAR-activatedcells in the presence of E7. Taken together, these results indicate thatE7 overcomes RafAR-induced arrest by abrogating the CDK2-inhibitoryfunction of p21^(Cip1).

(3) E7 Does Not Derepress p21Cip1-Associated CDK2 Activity

In response to observations that E7 maintains CDK2 activity in thecontext of p21^(Cip1)-mediated arrest, a model has been proposed thatp21^(Cip1)-associated CDK2 complexes are derepressed via thep21^(Cip1)-E7 interaction (Funk et al., 1997). However, others havereported that E7 does not associate with p21^(Cip1) (Hickman, E. S., S.Bates, and K. H. Vousden 1997. Pertubation of the p53 response by humanpapillomavirus type 16 E7 Journal of Virology. 71:3710-3718., Ruesch,M., and L. A. Laimins 1997. Initiation of DNA synthesis by humanpapillomavirus E7 oncoproteins is resistant to p21 mediated inhibitionof cyclin E-cdk2 activity Journal of Virology. 71:5570-5578.). Thismodel was examined within the context of the E7-RafAR system by testingtwo key predictions: (1) E7 should interact with p21^(Cip1) in celllysates, and (2) p21^(Cip1) should be associated with active cyclinE-CDK2 complexes. Standard coimrnmunoprecipitations with E7- andp21^(Cip1)-specific antibodies were used. No interaction between E7 andp21^(Cip1) was detected. However, E7 expression is low in this system,raising the possibility that the putative E7- p21^(Cip1) interaction wasnot detected due to technical limitations. To confirm thecoimmunoprecipitation results, purified recombinant GST-E7 was mixedwith control cell lysates and precipitated complexes were examined forthe presence of p21^(Cip1) by Western blot analysis. p21^(Cip1) was notfound in GST-E7 precipitates (FIG. 3A). As controls, p21^(Cip1) wasdetected in GST-cyclin E-CDK2 precipitates, and RB was associated withGST-E7. In the reciprocal experiment, GST-p21^(Cip1) was mixed with celllysate and an excess of radiolabelled E7. No p21^(Cip1)-E7 interactionwas observed, while GST-p130/³⁵S-E7 and GST-p21^(Cip1)/cyclin D1complexes were detected (FIG. 3B). These results indicate that E7 doesnot associate with p21^(Cip1) in this system.

In order to address the second corollary that p21^(Cip1) is associatedwith active cyclin E-CDK2 in E7-expressing cells, an immunodepletionapproach was used. Depletion of p21^(Cip1)-containing complexes fromcell lysates did not reduce the level of remaining cyclin E-CDK2activity. RafAR-induced E7-expressing cell lysates were subjected tothree rounds of immunodepletion with control or p21^(Cip1)-specificantibodies shown previously to precipitate all knowncyclin-CDK-p21^(Cip1) complexes (Cai and Dynlacht, 1998). p21^(Cip1) wasefficiently depleted as monitored by western blot analysis (FIG. 4A).Cyclin E-CDK2 complexes were then immunoprecipitated from depletedlysates and assessed for kinase activity. p21^(Cip1)-specificimmunodepletion reduced p21^(Cip1) levels by >95%, but did not alter theresidual cyclin E-CDK2 kinase activity (FIG. 4A), indicating that anydepleted p21^(Cpi1)-cyclinE-CDK2 complexes were inactive and that E7does not derepress p21^(Cip1)-associated CDK2. In addition, cyclin E andCDK2 are not rendered intrinsically resistant to p21^(Cip1) by E7, ascyclin E-associated kinase activity in lysates of control andE7-expressing cells was equally sensitive to inhibition by purifiedrecombinant GST-p21^(Cip1) (FIG. 4B).

(4) Enhanced Expression of Cyclin E Does Not Overcome RafAR-InducedArrest

Expression of E7 increased cyclin E protein levels approximately 2.5-3fold (FIG. 2A), which is consistent with the ability of E7 todysregulate RB-E2F transcriptional regulation of the cyclin E gene (Funket al., 1997; Zerfass et al., 1995). This effect is independent from,and additive to, the RafAR-induced elevation of cyclin E (FIG. 2A).Since E7 did not alter the intrinsic sensitivity of cyclin E-CDK2 top21^(Cip1), whether dysregulation of cyclin E expression by E7 may besufficient to overcome RafAR-induced, p21^(Cip1)-mediated arrest wasaddessed. This scenario was examined by stable expression of humancyclin E in RafAR-NIH3T3. Exogenous human cyclin E associates with andactivates endogenous murine CDK2 (Alevizopoulos et al., 1997; Vlach etal., 1996), as human cyclin E-specific antisera precipitated robustlevels of kinase activity (FIG. 5A, top panel). However, retroviralexpression of cyclin E did not abrogate RafAR-induced arrest (FIG. 5B).In addition, immunoprecipitation of human cyclin E complexes or totalendogenous CDK2 revealed that exogenous cyclin E expression did notprevent p21^(Cip1)-mediated inhibition of cyclin E-CDK2 (FIG. 5A),suggesting that the activity of E7 in this system is not defined solelyby induction of cyclin E levels. These observations are consistent withother reports that arrest imposed by Kip/Cip CKIs cannot be overcome byelevated physiological accumulation of cyclin E (Alevizopoulos et al.,1997; Perez-Roger et al., 1997).

(5) E7 Alters the Stoichiometry Between p21^(Cip1) and Cyclin E-CDK2

Upon RafAR-activation, cells expressing E7 maintained cyclin E-CD:K2activity (FIG. 2A) in complexes free of p21^(Cip1) (FIG. 4A), suggestingthat E7 may prevent the association of p21^(Cpi1) with a pool of cyclinE-CDK2 complexes. Equal amounts of p21^(Cpi1) were associated withcyclin E-immunoprecipitates in RafAR-induced control and E7-expressingcells (data not shown). However, since there is a significant increaseof cyclin E steady state levels in E7-expressing cells (FIG. 2A), thisobservation would imply that the stoichiometry of p21^(Cip1) and cyclinE-CDK2 is altered in the presence of E7. To illustrate this contentionmore clearly, cyclin E-containing complexes were immunoprecipitated fromRafAR-induced control or E7 cell lysate, standardizing on the level ofcyclin E expression (FIG. 6A, top panel). As seen in FIG. 6A (bottompanel), cyclin E-associated p21^(Cip1) was significantly lower inE7-expressing cells. This suggests that E7 expression may lead toaccumulation of p21^(Cip1)-free cyclin E-CDK2 complexes. In order toexamine this hypothesis more directly, RafAR-induced control or E7 celllysates were depleted with p21^(Cip1)-specific antisera as described inFIG. 4A. Mock or p21^(Cip1)-depleted lysates were subsequently analyzedfor remaining cyclin E, CDK2, and p21^(Cip1). The levels of cyclin E andCDK2 were significantly reduced by the p21^(Cip1)-specific antisera inboth cell types (FIG. 6B, left panel). This indicates that a substantialquantity of CDK2 complexes is associated with p21^(Cip1), consistentwith previous reports (Cai and Dynlacht, 1998; Zhang et al., 1994a;Zhang et al., 1994b). However, E7-expressing cell lysates retainedapproximately 3-fold more cyclin E and CDK2 than the controlcounterparts in p21^(Cip1)-depleted lysates. Since data from FIG. 4Ademonstrates that cyclin E-associated kinase activity is not associatedwith p21^(Cip1), the increased pool of p21^(Cip1)-free cyclin E-CDK2 inE7 cells (FIG. 6B) is likely responsible for E7-specific maintenance ofcyclin E-CDK2 activity during RafAR activation (FIG. 2B and FIG. 2C).Altogether, these observations indicate that the presence of E7 hindersp21^(Cip1) association with and inhibition of cyclin E-CDK2 complexes inRafAR-activated cells.

(6) E7 Prevents Raf-Induced p21^(Cip1) Nuclear Accumulation

Induction of p21^(Cip1) expression by p53 or other antimitogenic stimuliis accompanied by its nuclear accumulation. Since the localization ofp21^(Cip1) is considered to be important in its function as an inhibitorof proliferation (Goubin and Ducommun, 1995; Sherr and Roberts, 1995),the effects of RafAR on p21^(Cip1) cellular localization were examined.Upon staining with p21^(Cip1)-specific antibodies, a similar fraction(˜40%) of control and E7-expressing cells exhibited strong nuclearfluorescence (FIG. 7). This observation is consistent with reports thatp21^(Cip1) localizes to the nucleus during mid-G1 (Dulic et al., 1998)as 50-55% of asynchronous cells were in G1 (FIG. 1B). Interestingly,induction of RafAR resulted in a dramatic increase in p21^(Cip1) nuclearaccumulation, with >90% of control cells showing nuclearp21^(Cip1)-staining (FIG. 7). This observation suggests that Rafsignaling activates p21^(Cip1) function by regulating its cellularlocalization as well as increasing its synthesis. Notably, E7 expressionmarkedly reduced the RafAR-specific nuclear localization of p21^(Cip1)(FIG. 7), without altering induction of p21^(Cip1) expression (FIG. 2A).As cyclin E is primarily a nuclear protein (Ohtsubo et al., 1995), theseresults are consistent with the idea that E7 prevents p21^(Cip1)association with cyclin E-CDK2 by inhibiting RafAR-specific nuclearcompartmentalization of p21^(Cip1).

Since E7 has also been shown to abrogate the CKI function of the relatedp27^(Kip1) during cellular arrest (Schulze, A., B. Mannhardt, K.Zerfass-Thome, W. Zwerschke, and P. Jansen-Durr 1998.Anchorage-independent transcription of the cyclin A gene induced by theE7 oncoprotein of human papillomavirus type 16 Journal of Virology.72:2323-34.) (data not shown), it was examined whether mislocalizationwas a more general strategy by which E7 impinges on CKI function. TGF-βsignaling leads to p27^(Kip1)-mediated inhibition of cyclin E-CDK2 andcell cycle arrest in the epithelial cell line Mv1Lu (Reynisdottir, I.,K. Polyak, A. lavarone, and J. Massague 1995. Kip/Cip and Ink4 Cdkinhibitors cooperate to induce cell cycle arrest in response to TGF-betaGenes & Development. 9:1831-45.). It was investigated whether theeffects of E7 on p27^(Kip1) localization in the context of TGF-βutilizing an Mv1Lu derivative that expresses E7 in response todoxycycline (FIG. 15A). Consistent with previous observations (Demers,G. W., E. Espling, J. B. Harry, B. G. Etscheid, and D. A. Galloway 1996.Abrogation of growth arrest signals by human papillomavirus type 16 E7is mediated by sequences required for transformation Journal ofVirology. 70:6862-9.), E7 prevented the arrest imposed by TGF-β (datanot shown). Interestingly, TGF-β induced a robust nuclear accumulationof p27^(Kip1) that was alleviated in the presence of E7 (FIG. 15C andFIG. 15D). E7 altered p27^(Kip1) localization without affectingexpression levels of the CKI (FIG. 15B). It was also observed thismislocalization of p27^(Kip1) by E7 during growth factor deprivation infibroblasts (FIG. 12). These results suggest that E7 hinders Kip/Cip CKIfunction through a conserved mechanism (mislocalization) and in responseto multiple antimitogenic signals.

(7) The PI-3K/Akt Pathway is Required for E7-Mediated Abrogation ofRafAR-Induced Arrest

p21^(Cip1) contains a bipartite nuclear localization sequence (NLS) inits C-terminus (Goubin and Ducommun, 1995). Mutation of the NLS reducesthe capacity of p21^(Cip1) to inhibit CDK activity and cellularproliferation (Rossig et al., 2001; Sherr and Roberts, 1995; Zhou etal.,2001). Recently, Akt has been shown to phosphorylate threonine-145within the p21^(Cip1) NLS, leading to cytoplasmic localization ofp21^(Cip1) (Zhou et al., 2001). In accordance, inhibition of Akt resultsin reduced p21^(Cip1) phosphorylation, promoting nuclear accumulation ofp21^(Cip1) and growth arrest. In order to assess the role of Akt inE7-mediated abrogation of RafAR-induced arrest, RafAR was activated inE7-expressing cells in the presence or absence of LY294002, an inhibitorof the Akt-activator P13-K. As previously demonstrated, E7-expressingcells continue through the G1-S transition following RafAR activation.However, cell cycle progression of RafAR-induced, E7-expressing cellswas diminished in the presence of LY294002 (FIG. 8A). Incubation withLY294002 also restored Raf-induced nuclear accumulation of p21^(Cip1)(FIG. 8B), suggesting that PI3-K/Akt activity is required for E7 toimpair p21^(Cip1) localization and overcome RafAR-induced G1 arrest.Because inhibition of PI3-K could have pleiotropic effects beyond thespecific activity of Akt, the role of Akt in this system was furthershown by utilizing a dominant-negative mutant of Akt, Akt K179M.RafAR-activated control or E7-expressing cells were transientlytransfected with vector or Akt K179M expression plasmids in conjunctionwith a plasmid encoding the green fluorescent protein (GFP). DNAsynthesis of the transfected, GFP-positive cells was measured byBrdU-incorporation. As shown in FIG. 8C, introduction of Akt K179Mreduced BrdU incorporation of RafAR-induced E7-expressing cells to alevel similar to RafAR-induced control cells, indicating that Aktactivity is required for E7-mediated bypass of RafAR-arrest. Inaddition, Akt activity appears sufficient to rescue G1-S progressionduring RafAR-signaling, since transfection of RafAR-activated controlcells with a myristoylated, constitutively active form of Akt (myrAkt)restored BrdU incorporation to asynchronous levels (FIG. 8C). Theseresults suggest that Akt antagonizes Raf-induced arrest. Consequently,we examined the effects of RafAR signaling on the status of Akt usingantibodies which detect total or serine-473 phosphorylated (active) Akt.In control cells, the steady state levels of total and active Akt weredecreased upon RafAR-activation by 66% and 79%, respectively (FIG. 8D).This RafAR-induced reduction in Akt activity correlated with asignificant decrease in threonine-phosphorylated p21^(Cip1) despiteelevated levels of total p21^(Cip1) (FIG. 8E). In contrast,E7-expressing cells maintained total and activated Akt at or nearasynchronous levels and exhibited a modest increase inthreonine-phosphorylated p21^(Cip1) upon RafAR activation (FIG. 8D andFIG. 8E). Taken together, these observations suggest that RafARsignaling may converge on p21^(Cip1) in two ways: inducing transcriptionof p21^(Cip1) and stimulating p21^(Cip1) nuclear accumulation vianegative regulation of Akt. While E7 does not interfere withRafAR-specific expression of p21^(Cip1), the ability of RafAR to induceAkt down-regulation and p21^(Cip1) nuclear localization is prevented byE7, consistent with the idea that maintenance of Akt is important inE7-mediated cell cycle progression in the presence of RafAR-activation.This is further supported by analysis of the E7.C24G point mutationwithin the context of the RafAR system. Residue 24 resides in the LXCXEmotif of E7 and is essential for the E7-RB interaction (Barbosa et al.EMBO Journal. 9:153-60 (1990), Munger et al. EMBO Journal. 8:4099-105(1989). Importantly, it has been demonstrated that mutation of the LXCXEmotif disrupts the ability of E7 to cooperate with an activated Raspathway in cellular transformation (Banks et al. Oncogene. 5:1383-1389(1990), Edmonds et al. Virology. 63:2650-6 (1989), Phelps Journal ofVirology. 66:2418-27 (1992)). As represented in FIG. 9B, RafAR-NIH3T3cells stably expressing E7.C24G were as sensitive to RafAR-inducedinhibition of DNA synthesis as cells transduced with empty retrovirus,although the E7- and E7.C24G-expressing cell lines expressed comparablelevels of E7 as determined by Western blot analysis (FIG. 9A). Thissuggests that the LXCXE motif is essential in E7-mediation abrogation ofRafAR-induced arrest. In contrast to cells expressing wild-type E7,E7.C24G-expressing cells exhibited RafAR-induced loss of cyclinE-associated kinase activity and were incapable of maintaining activeAkt upon RafAR induction (FIG. 9C). Altogether, these observationsindicate that persistence of Akt activity may play a role in the abilityof E7 to abrogate p21^(Cip1), function and RafAR-induced arrest. That E7disrupts p21^(Cip1)-function without derepressing p21^(Cip1)-cyclin-CDK2complexes is relevant in multiple systems, including the host cell typeof HPV.

RafAR-activation led to a similar increase in p21^(Cip1) steady statelevels in control and E7-expressing cells. However, the stoichiometry ofcyclin E-associated p21^(Cip1) was lower in cells expressing E7 (FIG.6). The reduced association between p21^(Cip1) and cyclin E-CDK2 in thepresence of E7 correlated with an increased pool of p21^(Cip1)-freecyclin E-CDK2. As active cyclin E-CDK2 was not associated withp21^(Cip1) (FIG. 4), this elevation in inhibitor-free cyclin E-CDK2complexes is likely responsible for the E7-specific maintenance ofcyclin E-CDK2 activity during Raf signaling. E7-expressing cells alsoexhibited increased levels of steady-state cyclin E (FIG. 2), consistentwith observations that E7 disrupts E2F-RB-mediated transcriptionalregulation of the cyclin E gene (Martin et al., 1998; Zerfass et al.,1995). Although exogenous expression of cyclin E alone was insufficientto maintain cyclin E-CDK2 activity and cell cycle progression uponRafAR-activation (FIG. 5), E7-induced elevation in cyclin E synthesismay contribute to a net gain in p21^(Cip1)-free cyclin E-CDK2.Observations have been made with regard to the inyc proto-oncogene,where activation of Myc restores cyclin E-CDK2 activity in the presenceof elevated p21^(Cip1) or the closely related p27^(Kip1) (Bouchard etal., 1999; Perez-Roger et al., 1999; Perez-Roger et al., 1997; Rudolphet al., 1996; Steiner et al., 1995; Vlach et al., 1996). Myc-inducedcyclin E-CDK2 activity results from increased cyclin E and cyclin D1/D2expression, with cyclin D-CDK complexes sequestering Kip/Cip CKIs andcyclin E feeding a CKI-free pool of activatable cyclin E-CDK2 (Bouchardet al., 1999; Perez-Roger et al., 1999). As cyclins D1 and D2 are notupregulated upon E7 expression (FIG. 2), sequestration of p21^(Cip1) bycyclin D-CDK complexes does not likely account for the maintenance ofcyclin E-CDK2 activity in the RafAR-E7 system.

The results disclosed here establish a bi-directional antagonism betweenthese components of the Raf/MAPK and PI3-K/Akt signaling pathways. E7disrupts the effects of Raf on Akt (FIG. 8), and this correlates withcontinued phosphorylation and reduced nuclear localization of p21^(Cip1)in Raf-activated E7 cells (FIG. 7). The ability of E7 to preserve Aktactivity participates in E7's ability to overcome Raf-induced arrest,since chemical inhibition of the PI3-K/Akt pathway or introduction of adominant-negative Akt perturbs E7-mediated cell cycle progression in thepresence of Raf stimulation (FIG. 8). Indeed, expression of aconstitutively active form of Akt restored G1-S progression duringRaf-activation, underlining Akt as an important target in rescue fromRaf-induced arrest. An LXCXE-mutated form of E7 that could not preventthe Raf-induced effects on Akt was also impaired in its capacity toabrogate Raf arrest (FIG. 9). Since the LXCXE motif is essential for theinteraction between E7 and the RB tumor suppressor (Barbosa et al.,1990; Munger et al., 1989), the effects of E7 on Akt may involvedysregulation of RB-targeted genes.

Oncogenic activation of the Ras/Raf/MAPK pathway can lead to ap21^(Cip1)-dependent cell cycle arrest (Lloyd et al. Genes &Development. 11:663-77 (1997), Sewing et al. Molecular & CellularBiology 17:5588-97 (1997), Woods et al. Molecular & Cellular Biology17:5598-611 (1997)). HPV 16 E7 transforms primary cells in cooperationwith Ras and abrogates growth arrest elicited by various antimitogenicsignals that induce p21^(Cip1) expression (Demers et al. J. Virol.70:6862-9 (1996), Phelps et al. Cell. 53:539-47 (1988), Vousden et al.Oncogene Res. 3:167-75 (1988))). Disclosed herein, HPVI6 E7 ablates theCDK-inhibitory function of p21^(Cip1) in response to Raf activation byinhibiting its nuclear accumulation. Also disclosed is that Akt, aregulator of p21^(Cip1) localization, is required for the ability of E7to impair p21^(Cip1) nuclear accumulation and Raf-induced arrest. Theability to impinge on p21^(Cip1) function is conserved in severalRas-cooperating oncogenes. However, these oncogenes target p21^(Cip1) bydiffering mechanisms. For instance, SV40 Large T antigen interacts withand inactivates the p53 tumor suppressor, a transcriptional activator ofp21^(Cip1) (Dyson et al. Princess Takamatsu Symposia. 20:191-8 (1989),el-Deiry et al. Cell. 75:817-25 (1993)). In rat Schwann cells,expression of Large T antigen prevents Raf-induced, p53-mediatedexpression of p21^(Cip1), resulting in a mitogenic cellular response toRaf (Lloyd et al. 1997). Alternatively, other Ras-cooperating oncogenesdo not affect the expression or accumulation of p21^(Cip1). Mycactivates expression of factors that sequester p21^(Cip1) and theclosely related p27^(Kip1) (Perez-Roger et al. Eembo Journal. 18:5310-20(1999), Vlach et al. EMBO Journal. 15:6595-604 (1996)). In the contextof Raf-signaling, Myc restores cyclin E-CDK2 activity by inducingexpression of cyclin E and cyclin D2, with cyclin D-CDK complexessequestering p21^(Cip1) and cyclin E feeding a p21^(Cip1)-free pool ofactivatable cyclin E-CDK2 (Bouchard et al. EMBO Journal. 18:5321-33(1999), Perez-Roger et al. (1990)). Similar to Myc, E7 induces synthesisof cyclin E (FIG. 2A) (Funk et al. Genes & Development 11:2090-100(1997), Zerfass et al. J. Virol. 69:6389-99 (1995)). Increased levels ofcyclin E may elevate the pool of total cyclin E-CDK2 in E7-expressingcells, but, similar to previous observations (Perez-Roger et al.Oncogene. 14:2373-2381 (1997), is insufficient to bypass Raf-inducedarrest or restore cyclin E-CDK2 activity (FIG. 5A and FIG. 5B).

The results disclosed herein suggest a mechanism by which E7 preventsRaf-induced, p21^(Cip1)-mediated inhibition of cyclin E-CDK2. It isdisclosed that Raf-activation enhances the nuclear localization ofp21^(Cip1) (FIG. 7), and E7 expression reduces this Raf-specificp21^(Cip1) nuclear accumulation. Since p21^(Cip1) is thought toestablish a regulatory threshold that must be overcome for CDK2activation (Cai et al. PNAS USA 95:12254-9 (1998), Harper et al. Cell.6:387-400 (1995), Hengst et al. Genes & Development. 12:3882-8 (1998)),disclosed herein a reduction in nuclear p21^(Cip1) would effectivelylower the “local threshold” of p21^(Cip1) in the nuclear compartment.This is consistent with the disclosed observation that E7-expressingcells exhibited an increased pool of p21^(Cip1)-free cyclin E-CDK2despite Raf-induced elevation in p21^(Cip1) levels (FIG. 6A and FIG.6B).

Previously, a model was proposed in which E7 abrogates p21^(Cip1)function by directly interacting with and derepressingp21^(Cip1)-associated CDK2 complexes ( Funk, J. O., S. Waga, J. B.Harry, E. Espling, B. Stillman, and D. A. Galloway 1997. Inhibition ofCDK activity and PCNA-dependent DNA replication by p21 is blocked byinteraction with the HPV-16 E7 oncoprotein Genes & Development.11:2090-100., Jones, D. L., R. M. Alani, and K. Munger 1997. The humanpapillomavirus E7 oncoprotein can uncouple cellular differentiation andproliferation in human keratinocytes by abrogating p21Cip1-mediatedinhibition of cdk2 Genes & Development. 11:2101-11.). However, thedisclosed experiments do not support such a model. Interaction betweenE7 and p21^(Cip1) was not observed in the context of Raf signaling (FIG.3), and could not be detected in other cell types (data not shown)(Hickman, E. S., S. Bates, and K. H. Vousden 1997. Pertubation of thep53 response by human papillomavirus type 16 E7 Journal of Virology.71:3710-3718., Ruesch, M., and L. A. Laimins 1997. Initiation of DNAsynthesis by human papillomavirus E7 oncoproteins is resistant to p21mediated inhibition of cyclin E-cdk2 activity Journal of Virology.71:5570-5578.). In addition, immunodepletion experiments indicate thatp21^(Cip1)-associated cyclin E-CDK2 complexes are not active in thepresence of E7 (FIG. 4A), suggesting that E7 does not physicallyderepress p21^(Cip1)-cyclin E-CDK2 complexes. Furthermore, it was shownherein that the intrinsic sensitivity of cyclin E-CDK2 top21^(Cip1)-mediated inhibition is not altered in E7-expressing cells(FIG. 4B). Taken together, these data imply that E7 impinges onp21^(Cip1) function via an alternative mechanism as disclosed herein.

The ability of E7 to reduce nuclear accumulation of p21^(Cip1) andprevent inhibition of cyclin E-CDK2 suggests that p21^(Cip1)localization affects its regulation of CDK activity. Indeed, mutation ofthe nuclear localization sequence of p21^(Cip1) reduces its growthinhibitory function (Goubin et al. Oncogene. 10:2281-7 (1995), Zhou etal. Nature Call Biology. 3:245-52 (2001)). Subcellular location is animportant aspect of other CKI function. For instance, the coordinateinhibition of CDK4 and CDK2 by p15^(Ink4b) and p27^(Kip1) requiresproper compartmentalization of both CKIs within the cell (Reynisdottiret al. Genes & Development. 11:492-503 (1997)). It was also shown hereinthat E7 affects the nuclear localization of p27^(Kip1) upon serumwithdrawal (FIG. 12), indicating that the ability of E7 to abrogate thelocalization of Kip/Cip CKIs may be conserved in the context of otherbiological signals.

Akt has recently been shown to regulate p21^(Cip1) localization viaphosphorylation of its nuclear localization sequence (Zhou et al.(2001)). Interestingly, E7 prevents Raf-specific reduction in Akt levelsand p21^(Cip1) phosphorylation (FIG. 8D), suggesting that E7 alterslocalization of p21^(Cip1) by targeting Akt. In accordance, inhibitionof the PI(3)K/Akt signaling pathway abrogated the effects of E7 onp21^(Cip1) localization (FIG. 8B). Further experiments are required todetermine the role of Akt in E7-mediated bypass of otherp21^(Cip1)-associated arrest signals. However, Akt has been implicatedin diverse contexts including cell survival, proliferation, andtransformation, making Akt an intriguing putative target of E7 (Cofferet al. Biochemical Journal. 335:1-13 (1998), Datta et al. Genes &Development. 13:2905-27 (1999)).

Ras transduces extracellular information through a multitude ofsignaling cascades. Raf and Akt, components of two “parallel” Rassignaling pathways, have been shown to interact, with Aktphosphorylating and negatively regulating Raf activity (Rommel et al.Science. 286:1738-41 (1999), Zimmermann et al. Science. 286:1741-4(1999)). In this study, we provide evidence that Raf can functionallyantagonize Akt. We demonstrate that Raf can down-regulate steady-statelevels of total and active Akt (FIG. 8D). Importantly, transfection witha constitutively active Akt restored cell cycle progression during Rafsignaling (FIG. 8C), indicating that loss of Akt activity may beessential in Raf-induced arrest. Consistent with this, expression of E7maintained total and active Akt levels (FIG. 8C), and disruption of Aktactivity prevented E7-mediated bypass of Raf-arrest (FIG. 8A and FIG.8C). Interestingly, an E7 mutant deficient in the ability to bind anddisrupt RB does not maintain Akt activity (FIG. 9C), indicating that RBmay be involved in regulating Akt or factors that control Akt activity.Nevertheless, the observation that Raf down-regulates Akt functionestablishes precedent for bi-directional cross-talk between theseRas-effector pathways. The antagonism between Raf and Akt suggests thatthe cellular response elicited by simultaneous stimulation of Raf andAkt can be affected by the intensity and duration of the stimulation.For instance, anchorage detachment in primary fibroblasts leads toRaf-dependent anoikis (Zugasti et al. Molecular & Cellular Biology.21:6706-17 (2001)). Activation of Raf in this context requires loss ofAkt activity, and exogenously restored Akt activity can disrupt Rafactivation and restore cell survival. Likewise, sustained Akt activitycan prevent serum- and IGF-1-induced activation of the Raf/ERK signalingcascade, promoting myotube differentiation and hypertrophy (Rommel etal. (1999), Zimmermann (1999)). Disclosed herein, Raf activation leadsto loss of Akt activity and that this may be an essential event in thecellular arrest imposed by Raf.

Activation of the Ras/Raf/MAPK pathway can stimulate transformation orcellular arrest, depending on the signaling intensity and presence orabsence of cooperating oncogenic mutations (Hirakawa et al. PNAS USA.85:1519-23 (1988), Land et al. Molecular & Cellular Biology 6:1917-25(1986), Ruley et al., H.E. Cancer Cells. 2:258-68 (1990), Weinberg, R.A. O Cancer Research. 49:3713-21 (1989.), Woods et al. (1997)). It wasshown herein that the HPV16 E7 oncoprotein abrogates growth arrestimposed by Raf activity, a signal that elicits similar growth andmorphological phenotypes as Ras in primary and immortal cells (Lin etal. Genes & Development. 12:3008-19 (1998), Sewing et al. (1 997)).Consistent with these observations, E7 inhibits Ras-induced arrest andsenescence and subsequently transforms primary cells in cooperation withactivated Ras (Phelps et al. (1988), Vousden et al. (1988)). Raf-inducedarrest is dependent on the CKI p21^(Cip1) (Sewing et al. (1997), Woodset al. (1997)), and it was demonstrated herein that E7 impairsp21^(Cip1) CDK-inhibitory function during Raf signaling. As previouslymentioned, other oncogenes (e.g. Myc, SV40 Large T antigen) thatcooperate with Ras in cellular transformation also impair p21^(Cip1)function (Dyson et al. (1989), Perez-Roger et al. (1999)). Thisconvergence of function implies that inactivation of p21^(Cip1) may bean important mechanism by which oncogenic mutations alter the cellularresponse to the activated Ras/Raf pathway from growth arrest toproliferation. Indeed, genetic ablation of p21^(Cip1) confers aproliferative advantage in Ras-transduced fibroblasts and promotesaggressive Ras-induced epithelial tumorigenesis (Missero et al. Genes &Development. 10:3065-75 (1996), Sewing et al. (1997)). Theseobservations suggest that p21^(Cip1) is critical in suppressingRas-induced transformation.

2. Example 2 Materials and Methods

Cell culture. NIH3T3 cells stably expressing the RafAR fusion proteinhave been described previously (Sewing et al., 1997). This cell line andall derivatives were cultured in DMEM without phenol red (GIBCO-BRL)supplemented with 10% charcoal-stripped newborn calf serum (NCS,Hyclone). Cell lines stably expressing E7, E7.C24G, or human cyclin Ewere established via the recombinant amphotropic pBabe retroviral systemdescribed elsewhere (Morgenstern and Land, 1990). Upon infection, cellswere selected in 2.5 tig/mL puromycin (Sigma) and used for limitedgenerations. For all experiments, asynchronous cells were seeded at lowdensity (5×105 cells per 15 cm dish), and subsequently treated withRafAR-inducing 1.0 μM R1881 (methyltrienolone, Dupont) or vehicle(ethanol) for the indicated times. For TGF-β experiments, Mv1Lu cellswith doxycycline-inducible E7 expression, tet-E7 Mv1Lu (a kind gift fromM. O'Reilly), were treated with 3 ng/mL TGF-β with or without 2 μg/mLdoxycycline for 24 hours.

Cell cycle analysis. Cells were pulsed with 10 μM bromodeoxyuridine(BrdU) for 30 minutes, trypsinized, and fixed in 70% ethanol for 12hours at 4° C. Subsequently, cells were labeled with FITC-conjugatedα-BrdU (Boehringer Manheim), treated with RNase A (1 mg/mL, Sigma), andstained with propidium iodide (20 μg/mL, Sigma) following standardprotocols. Data was collected and analyzed by FACSCaliber (ELITE) andMulticycle software, respectively.

Immunoblotting and immunoprecipitations (IP). Cell pellets were lysed inHLB lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mMDTT, and protease inhibitor cocktail (Sigma P8340)) for 30 minutes withvortexing. After centrifugation, protein content was quantitated via astandard BioRad Bradford assay. For immunoblotting, 30-50 μg of celllysate was separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto nitrocellulose membrane(Schleicher and Schuell). After incubation with primary and secondaryantibodies, antigen detection was performed using the enhancedchemiluminescence kit from NEN. The following antibodies were obtainedfrom Santa Cruz: α-cyclin D1 (sc-450, Western), α-cyclin D2 (sc-593,Western), α-cyclin E (sc-481, IP and Western), α-human cyclin E (sc-198,IP and Western), α-CDK2 (sc-163 g, IP and Western), α-CDK4 (sc-260, IPand Western), α-p21^(Cip1) (sc-6246, Western), α-p21^(Cip1) (sc-397, IPand immunofluorescence), and a-phospho-threonine (sc-5267, Western).HPV-16 E7-specific and RB-specific antibodies were purchased from Zymedand Pharmingen, respectively. Antibodies recognizing Akt or Aktphosphorylated at serine-473 were obtained from Cell Signaling. Forimmunoprecipitations, cell lysates were incubated with primaryantibodies for 2 hours at 4° C., and immune-complexes were collected onProtein A-agarose beads (Santa Cruz) for an additional 1 hour. Thecomplexes were washed four times with HLB buffer and resolved by 12%SDS-PAGE for immunoblotting.

Kinase assays. After immunoprecipitation, immune-complexes were washedtwo additional times in kinase buffer (50 mM Tris, pH 7.5, 10 mM MgCl2,1 mM DTT). Kinase assays were performed in kinase buffer with 30 μM ATP,3 μCi [γ-32P]ATP, and 15 μg histone H1 (for cyclin E-CDK2 complexes) or2 μg GST-RB c-terminus (for CDK4 complexes) per reaction for 15 minutesat 23° C. Radiolabelled substrate was resolved on 12% SDS-PAGE andquantified with a phosphorimager and ImageQuant (Molecular Dynamics)software. For in vitro p21^(Cip1) inhibition experiments, purifiedrecombinant GST-p21^(Cip1) was incubated with 20 μg of target control orE7-expressing cell lysates for 30 minutes at 30° C. before assaying forcyclin E-associated histone H1 kinase activity as described above.

Plasmids and transfections. pBabe retroviral constructs expressing E7and E7.C24G were generated and are described elsewhere (Nead et al.,1998). The pBabe derivative expressing human cyclin E was describedelsewhere (Vlach et al., 1996). The pcDNA3 constructs encoding Akt K179Mwas described elsewhere (Burgering and Coffer, 1995) and myristoylatedAkt (myr-Akt) (Kohn et al., 1996) was described elsewhere. TheGFP-expression plasmid, pEGFPC1, is commercially available (Clontech).For transient transfections, control or E7-expressing cells were seededon 60 mm dishes at a density of 1.5×10⁵ cells/plate. Cells weretransfected 24 hours later with the indicated plasmids usingLipfectAmine 2000 (Invitrogen) according to manufacturer's instructions.After a 5 hour incubation, the transfection medium was replaced withDMEM without phenol red (GIBCO-BRL) supplemented with 10%charcoal-stripped newborn calf serum (NCS, Hyclone) for an additional 6hours. Subsequently, cells were treated with vehicle (ethanol) or R1881for an additional 30 hours, pulsed with 10 μM BrdU during the last 10hours of treatment, and analyzed for BrdU incorporation byimmunofluorescence (see below).

Immunofluorescence. For p21^(Cip1) localization experiments, cells wereplated in six-well dishes and treated with R1881 or vehicle. Aftertreatment, cells were fixed in 3.7% paraformaldehyde, perneabilized in0.2% Triton X-100 and 10% FBS in PBS for 10 minutes, and incubated withp21^(Cip1)-specific antibodies (1:100 dilution) or normal IgG for 12hours at 4° C. Cells were washed three times in PBS with 10% FBS andstained with fluorophore-conjugated α-rabbit (1:200 dilution, MolecularProbes) for 30 minutes. All antibody solutions were prepared in 10% FBSin PBS. For transfection experiments, cells were pulsed with BrdU for 10hours prior to fixation. Cells were fixed and permeabilized as describedfor p21^(Cip1) localization experiments, and cellular DNA was denaturedby incubation with DNase I (100 U/mL, Gibco) for 1 hour at 37° C.BrdU-positive cells were stained with fluorophore-conjugated α-BrdU(Molecular Probes) for 12 hours at 4° C. Antigen staining was visualizedby inverted fluorescence microscopy (Olympus CK40), and images werecaptured with Quality Imaging camera and software. Exposure times werekept constant for each experiment.

3. Example 3

a) Inactivation of Akt by c-Raf-1 is Rapid:

c-Raf-1 was induced by addition of the androgen analog R1881, asdescribed herein and the cells harvested at various times thereafter. Ascan be seen from FIG. 13, the down-regulation of the active Akt (P-Akt)is observed within 5 hours post-treatment, while total protein levelsonly decrease at about 19-26 hours post-treatment. As discussed inExample 1, the cell cycle arrest occurs about 20 hours post-treatment,and is consistent with accounting for the reduced levels ofnon-phosphorylated-inactive Akt. Thus, while total amounts of Akt do notchange at the early time points the phosphorylated active formdisappears early on. The results indicate that the down-regulation ofactive Akt is c-raf-1 mediated and not a result of cell cycle arrest.

b) c-Raf-1 Inhibits Akt Activity Through MEK1:

The down-regulation of activie Akt (P-Akt) on induction of c-Raf-1requires MEK1, which is a down stream target of cRaf-1. This wasdetermined by adding a MEK1 inhibitor (U0126) at the same time as theandrogen analog, R1881 (FIG. 14, left hand panel, lanes 5 and 6). In thepresence of the inhibitor active Akt down-regulation is inhibited (FIG.14, compare lanes 1 and 2 with 5 and 6 in left panel). In fact, in thepresence of the inhibitor the basal level of active Akt is increased,consistent with MEK1 normally controlling Akt activity (FIG. 14, comparelanes I and 5).

c) New Protein Synthesis is Required for c-Raf-1-DependentDown-Regulation of Akt Activity:

To determine if new protein synthesis is required for thedown-regulation of Akt, the cells were treated with cyclohexamide (aprotein synthesis inhibitor, which acts on ribosomes) was added alongwith R1881. As can be seen from FIG. 14, (compare lanes 1 and 2 withlanes 3 and 4 in left panel), cyclohexamide treatment (CXM) inhibitedthe down-regulation of Akt. Therefore, new protein synthesis is requiredfor the down-regulation of Akt by c-Raf-1.

d) Proteosome Activity Not Required for the Down-Regulation of AktActivity:

Since there is a reduction in the active form of Akt (P-Akt) within 5hours, it was determined if this Akt component was degraded by theproteosome. Using the proteosome inhibitor, MG132, we showed thatdown-regulation of P-Akt was similar to control cells (FIG. 14 comparelanes 1 and 2 with 5 and 6, right panel). Therefore, the active form ofAkt is not degraded.

These results indicate that Akt is de-phosphorylated by a phosphataseprotein controlled by c-Raf-1.

e) E7 Impairs DNA Damage-Induced Nuclear Localization of p21^(Cip1) inHFK.

DNA damage was used to arrest cells through adriamycin treatment.Control- or E7-expressing HFK were treated with vehicle or 0.1 mMAdriamycin for 17 hours and pulsed for 1 hour prior to fixation in 4%paraformaldehyde. Subsequent to fixation, chromatin was denatured with2N HCl, and cells were permeabilized with 0.2% Triton X-100, and stainedwith AlexaFluor 588-conjugated α-BrdU. Cells were scored forBrdU-positive nuclei. The number of cells with BrdU-positive nuclei isrepresented as a percentage of total cells counted. FIG. 16 A shows theaverage and standard deviation values shown are from two independentexperiments with at least 300 cells counted per experiment.

In FIG. 16B cells were treated as in FIG. 16A and in addition werestained with p21^(Cip1)-specific antibody and scored for nuclear orwhole-cell staining. The number of cells with nuclear p21^(Cip1)accumulation is represented as a percentage of total cells counted. FIG.16B shows the average and standard deviation values shown are from twoindependent experiments with at least 200 cells counted per experiment.In FIG. 16C cell lysates were isolated at the same time as in FIG. 16Aand Western blots carried out for active Akt and (FRET), in the presenceof the putative inhibitor and the in absence of the putative inhibitor,wherein a decrease in FRET in the presence of the putative inhibitor ascompared to FRET measurement in its absence indicates the putativeinhibitor inhibits binding between the two molecules. This type ofmethod can be performed with a cell system as well. Thus, for example,putative inhibitors could be attached to a fluorescence acceptor, andAkt could be attached to a fluorescence donor, and then in the disclosedsystems, Akt activity monitored and correlated with the moleculesinteracting with Akt to identify molecules inhibiting Akt activitythrough interaction with Akt. It would also be possible to detect BrDUuptake in cells in a high throughput screen. BrDU would be taken up incells that were not cell cycle arrested and so would indicate a drugtreatment did not work.

Disclosed are cells comprising, a) a regulatable nucleic acid comprisingsequence encoding a Raf gene and b) a nucleic acid comprising sequenceencoding an E7 gene.

Disclosed are cells comprising, a regulatable nucleic acid comprisingsequence encoding a Raf gene and sequence encoding an E7 gene.

Disclosed are cells comprising, a) a regulatable nucleic acid comprisingsequence encoding a Raf gene and b) a nucleic acid comprising sequenceencoding a cyclin gene.

Also disclosed are cells comprising, a regulatable nucleic acidcomprising sequence encoding a Raf gene and sequence encoding a cyclingene.

Disclosed are cells further comprising an inhibitor of Akt, an inhibitorof E7, a potential inhibitor of Akt degradation, and/or a potentialinhibitor of E7 activity. Also disclosed are cells further comprising aninhibitor of PI3K.

These systems can be used to identify compositions having the desiredeffects on E7 and Akt described herein. For example, compositions whichpotentially inhibit the effect of E7 described herein can be assayed fortheir effect in the system, for example, those that promote p21^(Cip1)nuclear transport, through, for example, inhibiting E7 promotedmaintenance of Akt. The systems can be used in a variety of ways asdiscussed herein.

4. Sequence Similarities

It is understood that as discussed herein the use of the terms homologyand identity mean the same thing as similarity. Thus, for example, ifthe use of the word homology is used between two non-natural sequencesit is understood that this is not necessarily indicating an evolutionaryrelationship between these two sequences, but rather is looking at thesimilarity or relatedness between their nucleic acid sequences. Many ofthe methods for determining homology between two evolutionarily relatedmolecules are routinely applied to any two or more nucleic acids orproteins for the purpose of measuring sequence similarity regardless ofwhether they are evolutionarily related or not. p21^(CIP) levels beforeand after adriamycin treatment. FIG. 16 shows that 21^(CIP) is increasedafter adriamycin treatment in both control and E7-expressing HFK andthat active Akt levels are maintained only in E7-expressing cells.

F. REFERENCES

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G. SEQUENCES

1. SEQ ID NO:1 Protein hormone binding domain of human androgen receptor(aa 646-917)

2. SEQ ID NO:2 Protein c-Raf-1 (aa 305-648)

3. SEQ ID NO:3 K-ras (aa 169-188)

4. SEQ ID NO:4 human p21^(Cip1) protein sequence

5. SEQ ID NO:5 human p21^(Cip1) DNA sequence

6. SEQ ID NO:6 human Akt/PKB protein sequenc

7. SEQ ID NO:7 human Akt1 DNA sequence

8. SEQ ID NO:8 HPV16 E7 aa sequence

9. SEQ ID NO:9 HPV16 E7 DNA sequence

10. SEQ ID NO:10 HPV16 E7 degenerate DNA sequence 6^(th) nucleotidechanged from T to a C at capital C, still encoding His

11. SEQ ID NO:11 HPV16 variant E7 aa sequence I to V at position 38

12. SEQ ID NO:12 DNA encoding HPV16 E7 variant set forth in SEQ ID NO:11(A to G change at capital G)

13. SEQ ID NO:13 degenerate DNA encoding SEQ ID NO 11. T to C change atcapital C still encoding His

14. SEQ ID NO:14, Accession No. AAA42001, raf protein, rattus norvegicus

15. SEQ ID NO:15, Accession No. X03484 human mRNA for raf oncogene

16. SEQ ID NO:16, Accession No. AAA28142 raf proto-oncogene,Caenorhabditis elegans

17. SEQ ID NO:17, Accession No. NM-076862 Caenorhabditis elegans MAPkinase kinase or Erk Kinase MEK-1 DNA

18. SEQ ID NO:18, Accession No. P53349, mitogen-activated protein kinasekinase kinase 1 (MAPKK1) (Erk activator kinase 1) (MAPK/ERK kinasekinase 1) (MEK1 kinase 1) (MEKK1), mus musculus

19. SEQ ID NO:19, Accession No. Q02750 Dual Specificitymitogen-activated protein kinase kinase 1 (MAP kinase kinase 1) (MAPKK1)(Erk activator kinase 1). (MAPK/ERK kinase 1) (MEK1), homo sapiens

20. SEQ ID NO:20, Accession No. P31938, Dual Specificitymitogen-activated protein kinase kinase 1 (MAP kinase kinase 1) (MAPKK1)(Erk activator kinase 1) (MAPK/ERK kinase 1) (MEK1), mus musculus

21. SEQ ID NO:21, Accession No. NP-002746 mitogen-activated proteinkinase kinase 1, protein kinase, mitogen activated, kinase 1 (MAP kinasekinase 1) homo sapiens

22. P04049 RAF proto-oncogene serine/threonine-protein kinase (RAF-1)

1. A method of identifying a compound that inhibits E7 cellularproliferation activity comprising, a) administering a compound to asystem, wherein the system maintains Akt activity; b) assaying theeffect of the compound on the amount of Akt activity in the system; andc) selecting a compound which causes a decrease in the amount of Aktactivity present in the system.
 2. The method of claim 1, wherein thesystem comprises an arrest signal.
 3. The method of claim 1, wherein thearrest signal comprises an inducible Raf protein or conserved variant ofthe Raf protein.
 4. The method of claim 3, wherein the inducible Rafprotein is cRaf-1 or a conserved variant of cRaf-1.
 5. The method ofclaim 1, wherein the step of assaying the effect of the compoundcomprises using an antibody for Akt.
 6. A method of inhibiting E7cellular proliferation activity comprising administering a compound,wherein the compound decreases the amount of Akt activity, wherein thecompound is defined as a compound capable of being identified byadministering the compound to a system, wherein the system maintains Aktactivity, assaying the effect of the compound on the amount of Aktactivity in the system, and selecting a compound which causes a decreasein the amount of Akt activity present in the system.
 7. A method ofinhibiting E7 cellular proliferation activity comprising administering acompound that decreases the amount of Akt activity.
 8. A method ofmaking a composition capable of inhibiting E7 cellular proliferationactivity comprising mixing an E7 inhibiting compound with apharmaceutically acceptable carrier, wherein the compound can beidentified by administering the compound to a system, wherein the systemmaintains Akt activity, assaying the effect of the compound on theamount of Akt activity in the system, and selecting a compound whichcauses a decrease in the amount of Akt activity present in the system.9. A method of making a compound that inhibits E7 cellular proliferationactivity comprising, a) administering a compound to a system, whereinthe system causes maintenance of Akt activity, b) assaying the effect ofthe compound on the amount of Akt activity in the system, c) selecting acompound which causes a decrease in the amount of Akt activity presentin the system, and d) synthesizing the compound.
 10. A method ofidentifying a compound capable of reversing the effect E7 has on Aktcomprising, a) administering a compound to a system, wherein the systemcomprises E7 maintenance of Akt activity, b) assaying the effect of thecompound on E7 maintenance of Akt activity, and c) selecting a compoundwhich inhibits E7 maintenance of Akt activity.
 11. A method ofinhibiting E7 cellular proliferation activity comprising administering acompound, wherein the compound is identified as decreasing Akt activity.12. A method of inhibiting E7 cellular proliferation activity comprisingadministering an inhibitor of E7 maintenance of Akt activity, whereinthe inhibitor is a compound capable of being identified by administeringthe compound to a system, wherein the system comprises E7 maintenance ofAkt activity, assaying the effect of the compound on E7 maintenance ofAkt activity, and selecting a compound which inhibits E7 maintenance ofAkt activity.
 13. A method of inhibiting E7 cellular proliferationactivity comprising administering an inhibitor of E7 maintenance of Aktactivity.
 14. A method of making a composition capable of inhibiting E7maintenance of Akt activity comprising mixing the compound with apharmaceutical carrier and wherein the compound can be identified byadministering the compound to a system, wherein the system comprises E7maintenance of Akt activity, assaying the effect of the compound on E7maintenance of Akt activity, and selecting a compound which inhibits E7maintenance of Akt activity.
 15. A method of making a compound capableof reversing the effect E7 has on Akt comprising, a) administering acompound to a system, wherein the system comprises E7 maintenance of Aktactivity, b) assaying the effect of the compound on E7 maintenance ofAkt activity, c) selecting a compound which inhibits E7 maintenance ofAkt activity, and d) synthesizing the compound.
 16. A method ofinhibiting E7 cellular proliferation activity comprising administering acompound, wherein the compound is identified as inhibiting E7maintenance of Akt activity.
 17. A method of identifying a compoundwhich promotes the nuclear localization of p21^(Cip1) comprising, a)administering a compound to a system, wherein the system comprises E7p21^(Cip1) cytoplasmic localization activity, b) assaying the effect ofthe compound on E7 p21^(Cip1) cytoplasmic localization activity, and c)selecting a compound which promotes p21^(Cip1) nuclear localizationactivity.
 18. A method of promoting p21^(Cip1) nuclear localization,comprising a) administering a compound to a system, wherein the systemcomprises E7 p21^(Cip1) cytoplasmic localization activity, b) assayingthe effect of the compound on E7 p21^(Cip1) cytoplasmic localizationactivity, and c) selecting a compound which promotes p21^(Cip1) nuclearlocalization activity.
 19. A method of identifying an inhibitor of aninteraction between Akt and E7 comprising a) administering a compound toa system, wherein the system comprises E7, b) assaying the effect of thecompound on an E7-Akt interaction, and c) selecting a compound whichinhibits E7 Akt interaction.
 20. A cell comprising, a) a regulatablenucleic acid comprising sequence encoding Raf or conserved variant, andb) a nucleic acid comprising sequence encoding an E7 or conservedvariant.
 21. The cell of claim 21, wherein the Raf is cRaf-1 orconserved variant.
 22. A cell comprising, a) a regulatable nucleic acidcomprising sequence encoding Raf or conserved variant and sequenceencoding E7 or conserved variant.
 23. The cell of claim 20 furthercomprising an inhibitor of Akt.
 24. The cell of claim 20 furthercomprising and inhibitor of E7.
 25. The cell of claim 20 furthercomprising an inhibitor of PI3K.
 26. A cell comprising, a) a regulatablenucleic acid comprising sequence encoding Raf or conserved variant andb) a nucleic acid comprising sequence encoding cyclin or conservedvariant.
 27. A cell comprising, a) a regulatable nucleic acid comprisingsequence encoding Raf or conserved variant and sequence encoding cyclinor conserved variant.
 28. A method of inhibiting aberrant cellularproliferation comprising, administering a compound which inhibits E7maintenance of Akt activity.
 29. The method of claim 26, whereinadministering the compound occurs in a subject.
 30. The method of claim27, wherein the subject is a subject who has cancer.
 31. A method ofinhibiting E7 cellular proliferation activity, comprising administeringa compound that promotes or maintains MEK-1 activity.